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TEXT-BOOK

OF

PHYSIOLOGICAL CHEMISTRY

IN THIRTY LECTURES

EMIL ABDERHALDEN

O. PROFESSOR Fl'R PHVSIOLOGIE DES PHYSIOLOGISCHEN INSTITUTS

DEH TIEHARZTLICHEN HOCHSCHUI.E BERLIN UND

ITNIVERSITATS-PROFESSOR

TRANSLATED BY

WILLIAM T. HALL

IXSTRUCTOR IK CHESnSTRV, MASSACHUSETTS INSTITUTE
OF TECHNOLOGY

AND

GEORGE DEFREN

FIRST EDITION

FIRST THOUSAND

NEW YORK

JOHN WILEY & SONS

London: CHAPMAN & HALL, LmrraD

1908

Copyright, 1908,

WILLIAM T. HALL

Stanbope ]prcs5

AUTHOR'S PREFACE.

The following lectures are not intended to embrace all the results of
physiological-chemical investigation. On the contrary, the aim has
been to discuss only those discoveries which are known to be of general
interest and importance. .\11 isolated facts, of however great significance
for the inilividual investigator in the field, of which the value is not
fully established and where the connection to other observations is not
clearly known, have been intentionally omitted. The lectures should
lead to individual thought and serve to incite further investigation and
at the same time give one a general survey of the field covered by physi-
ological chemistry. Corresponding to this purpose, great care has been
used with regard to selecting the literature upon which the lectures are
based. It is evident that within the space allotted only a limited
number of researches could be cited. The " Zentralbliitter " with their
abstracts and especially Asho and Spiro's " Ergebnisse der Physiologie "
must serve to fill in the gaps. Methods and descriptions of individual
comfKJunds are not discussed in detail. It is not possible for any one to
work well from brief descriptions. Practical laboratory experience is
necessary, and cannot be replaced by anything else. The reader is
referred to Felix Hoppe-Seyler's " Handbuch der physiologisch-chemi-
schen Analyse " for all such particulars.

E. ABDERHALDEN.

TRANSLATOR'S PREFACE.

One of the chief difficulties which arose in the preparation of
this transhxtion was with regard to the proper spelHng of the com-
pounds mentioned. Many of them have not been described much in
English, so that English-speaking scientists are often better acquainted
with the German orthography. Some of the chemical compounds have
been spelletl in three different ways by writers of good English. This
would almost lead one to believe that there is no good authority in
English for the spelling of chemical names. Many writers have followed
the German spelling as nearly as possible in describing compounds which
have hitherto been mentioned only in German literature. This must
necessarily lead to confusion, particularly because the ending e in German
usually signifies the plural, whereas it does not in English. The Chemical
Society of London in its Abstracts has mentioned nearly every sub-
stance touched upon in this book and has adopted certain rules for
spelling which its abstractors are required to follow. These rules have,
in the main, been adopted by the American Chemical Society and the
American Chemical Journal. According to these rules, -^ (1) All hy-
droxy! derivatives of hydrocarbons should end in ol, thus glycerol,
resorcinol and mannitol rather than glycerine, resorcin and mannite.
(2) Compounds which are not alcohols and have names ending in ol
should be written ole, as anisole, indole. (3) When a substituent is one of
the groups NH,, NHR, NR,, NH or NR its name should end in ine, thus
aminopropionic acid and not amidopropionie acid. (4) The ending ine
should be reserved for these basic substances, as aniline instead of anilin,
and the termination in should be reserved for glycerides, glucosides,
bitter principles and proteins, such as palmitin, amygdalin and albumin.
It seems to us that these are the best rules for English-speaking chemists
to follow at present. The rules cited are those which pertain particu-
larly to the substances described in these lectures.

In two cases we have intentionally deviated from the practice of the
above-mentioned chemical journals. We have used the word ferment to
designate "that which is capable of causing fermentation" (Century Dic-
tionary) and have not attempted to distinguish between ferments and
enzymes. This distinction was based upon an error, as Buchner has so
positively shown, and has led to much confusion. Again, we have fol-
lowed the author rather than the chemical journals with regard to the

vi TRANSLATOR'S PREFACE.

use of the words protein and proteid. While this book was in press the
"Joint Recommendations of the Committees on Protein Nomenclature"
was published in Science, in which the first recommendation was that the
word proteid should be abolished. They would call what Dr. Abder-
halden designates as proteids the conjugated proteins. The confusion
with regard to the word proteid has arisen from the fact that some writers
have designated as proteids the whole protein group, while others have
used the word only for these compound proteins. It was too late to
adopt the recommended nomenclature, as many of the plates were already
cast. It seems probable, however, in view of the rapid progress which is
now being made in this branch of chemistry that before long we shall be
able to adopt a chemical classification of the proteins which shall be
better than any yet proposed.

It has not seemed best to give all of the titles to the papers cited in
the footnotes. Most of these titles which appear in the original lectures
are in German, and in some cases they were evidently taken from the
Centralblatt and are German translations of English titles. It seemed
sufficient to give merely the abbreviated titles of the journals where the
references could be found with the volume and page. The abbreviations
used are, in the main, those adopted by the American Chemical Society
in their Chemical Abstracts, the principal ones being given in the front of
this book.

Dr. Abderhalden has kindly looked over all of the "page proof" and
has suggested numerous changes bringing the literature in some cases up
to 1908. Professor F. Jewett Moore of the Massachusetts Institute of
Technology as well as Dr. Percy G. Stiles of Simmons College and the
Institute of Technology have also read all of the proof and have rendered
invaluable aid by their many suggestions and criticisms. If this trans-
lation meets with the same friendly reception that has been accorded to
the original, credit is due fully as much to each of these two gentlemen
as to either one of the translators.

WILLIAM T. HALL.

Massachusetts In|titdte of Technologt,
July 11, 1908.

ABBREVIATIONS USED IN FOOTNOTES.

ABBREVIATED TITLE. FULL TITLE.

AUgem. med. Zentr AUgemeine medizinische Zentralzeitung.

Am. Chem. J American Chemical Journal.

Am. J. Physiol American Journal of Physiology.

Ann. Phil Liebig's Annalen der Chemie.

Ann. chim. phys Annalt^s de chimie et de physique.

Ann. de chim Annales de chimie.

Ann. inst. Pasteur Annales de I'in.stitute Pasteur.

Ann. Phil Annals of Philosophy.

Arch. Anat. Physiol Archiv fur Anatomie und Physiologie.

Arch, exper. Path. Pharm Archiv fiir experimentelle Pathologic und Pharma-

kologie.

Arch, exper. Path. Therapie Archiv fur experimentelle Pathologie und Therapie.

Arch. Gynak Archiv fiir Gynakologie.

Arch. Hyg Archiv fiir Hygiene.

Arch. Kinderheilk Archiv fiir Kinderheilkunde.

Arch. klin. Med Archiv fiir klinische Medizin.

Arch. path. Anat Archiv fiir pathologische Anatomie und Physiolo-
gie und fiir klinische Medizin.

Arch. Pharm Archiv der Pharmacie.

Arch, physiol. Ges Archiv der physiologischen Gesellschaft.

Beitr Beitrage. See Hofraeister.

Ber Berichte der deutschen chemischen Gesellschaft.

Ber. Berl. Akad. Wissensch Berichte der Berliner .\kademie der Wissenschaften.

Ber. deut. hot. Ger Berichte der deutschen botanischen Gesellschaft.

Berl. klin. Wochschr Berliner klinischer Wochenschrift.

Biochem. Zentr Biochemischer Zentralblatt.

Bot. Ztg Botanische Zeitung.

Brit. Med. J British Medical Journal.

Bull. soc. chim Bulletin de la soci^t^ chimique de Paris.

Centralbl Chemischer Centralblatt.

Compt. rend Comptes-rendus hebdomadaires des stances de I'aca-

d^mie des sciences.
Compt. rend. soc. biol Comptes-rendus hebdomadaires des stances et

memoirs de I'acad^mie des sciences.

Deut. Arch. klin. Med Deutsche Archiv fiir klinische Medizin.

Deut. med. Wochschr Deutsche medizinische Wochenschrift.

Ergeb. Physiol. (Asherand Spiro).Asher and Spire's Ergebnisse der Physiologie.

Hofmeister's Beitr Beitrage zur chemischen Physiologie und Pathologie.

viii ABBREVIATIONS USED IN FOOTNOTES.

ABBBEVIATED TITLE. FULL TITLE.

J. Anat. Physiol Journal of Anatomy and Physiology.

J. Exper. Med Journal of E.xperimental Medicine.

J. Chem. Soc Journal of the Chemical Society, London.

J. pharm. chim Journal de pharmacie et de chimie.

J. Physiol The Journal of Physiology.

J. pr. Chem Journal fiir praktische Chemie.

Med. Klinik Medizinische Klinik.

Mem. couronn. acad. roy. Belg. . .M6moirescouronn6s de I'aead^mie royale de Belgique.

Monatsh Monatshefte fiir Chemie und verwandte Telle der

Wissenschaften.
Miinch. med. Wochschr Miinchener medizinische Wochensohrift.

PfJiiger's Arch Pfliiger's Archiv fiir die gesammte Physiologie des

Menschen und der Tiere.

Pharm. Zentr Pharmazeutische Zentralblatt.

Phil. Trans. Roy. Soc Philosophical Transactions of the Royal Society of

London.

Pr. Chem. Soc Proceedings of the London Chemical Society.

Pr. Physiol. Soc Proceedings of the Physiological Society.

Pr. Roy. Soc Proceedings of the London Royal Society.

Pr. Roy. Soc. Edinburgh Proceedings of the Edinburgh Royal Society.

Sitzber. Akad. Wiss. Berl Sitzungsberichte der konigliche Preussischen Aka-

demie der Wissenschaften zu Berlin.

Sitzber. Akad. Wiss. Wien Sitzungsberichte der konigliche Akademie der Wis-
senschaften zu Wien.

Sitzber. Gesel. Morph. u. Physiol. Sitzungsberichte der Gesellschaft fiir Morphologic
Miinchen und Physiologie in Mimchen.

Sitzber. kgl. Gesel. Wiss. Upsala. .Sitzungsberichte der koniglichen Gesellschaft der
Wissenschaften zu Upsala.

Sitzber. physikal. med. Gesel. Sitzungsberichte der physikalisch-medische Gesell-
Wurzburg schaft zu Wurzburg.

Sitzber. Miinchener Akad Sitzungsberichte der koniglichen bayerischen Aka-
demie der Wissenschaften zu Miinchen.

Skand. Arch. Physiol Skandinavisches Archiv fiir Physiologie.

Trans. Chem. Soc Transactions of the Chemical Society.

Verb. Ges. Naturforsch. Aerzte. . .Verhandlung der Gesellschaft deutscher Naturfor-

scher und Aerzte.
Virchow's Arch Archiv fiir pathologische Anatomic und Physiologie

und fiir kHnische Medizin.

Z. allg. Biol Zeitschrift fiir allgemeine Biologic.

Z. allg. Physiol Zeitschrift fiir allgemeine Physiologie.

Z. Biol Zeitschrift fiir Biologic.

Z. Elektrochem Zeitschrift fiir Elektrochemie.

Z. exper. Path. Therap Zeitschrift fiir e.xperimentelle Pathologic und

Therapie.

ABBREVIATIONS USED IN FOOTNOTES. ix

ABBREVIATED TITLE. FULL TITLE.

Z. Hyg Zeitschrift fur Hygiene und Infektionskrankheiten.

Z. innere Med Zeitschrift fvir innere Medizin.

Z. klin. Med Zeitschrift fiir klinische Medizin.

Z. physikal. Chem Zeitschrift fiir physikalische Chemie.

Z. physiol. Chem Hoppe-Seyler's Zeitschrift fiir physiologischen

Chemie.

Z. rat. Med Zeitschrift fiir rationelle Medizin.

Zenlr. Bakt. u. Parasitienkunde . . Zentralblatt fiir Bakteriologie und Parasitienkunde.

Zentr. med. Wissensch Zentr.alblatt fiir die niedizinisohe Wissenschaften.

Zentr. Physiol Zentralblatt fiir Physiologic.

Zentr. Stoffwechs. Verdauungs- Zentralblatt fiir Stoffwechsel und Verdauungskrank-
krankheit heiteu.

TABLE OF CONTENTS.

LECTURE I

PAOE

Introduction 1

LECTURE II

Carbohydrates. I. In General — Monosaccharides — Glucosamine —

Glucuronic Acid 13

LECTURE III
Carbohydrates. II. Polysaccharides 36

LECTURE IV

Carbohydrates. III. Metabolism of Carbohydrates in Plant and Animal

Organisms 50

LECTURE V

Carbohydrates. IV. Building up and Breaking Down of Carbohydrates

in the Animal Organism 76

LECTURE VI
Fats — Lecithin — Cholesterol 101

LECTURE VII

Albumins or Proteins. I. Elementary Composition — Simple Substances

OR Mixtures — Classification 119

LECTURE VIII
Albumins or Proteins. II. The Components of Protein 146

LECTURE IX

Albumins or Protei.vs. III. Composition of Individual Proteins — Con-

STITITTION 171

xii TABLE OF CONTENTS.

LECTURE X

PAGE

Albumins or Proteins. IV. Degradation and Formation of Protein in

THE Animal and Vegetable Organisms 193

LECTURE XI

Albumins or Proteins. V. Decomposition of Protein in the Tissues — The

End-products op Albumin Metabolism 221

LECTURE XII
Albumins or Proteins. VI. Metabolic End-products 251

LECTURE XIII ,
The Nucleoproteids and their Cleavage-products 275

LECTURE XIV
Mutual Relations between Fats, Carbohydrates, and Albumins. 1 301

LECTURE XV

Mutual Relations between Fats, Carbohydrates, and Albumins. II. Law

OF Isodynamics 331

LECTURE XVI

Inorganic Foods. I. Importance of Inorganic Substances as Building

Material of the Cells and Tissue. — Water, Salts 349

LECTURE XVII
Inorganic Foods. II 380

LECTURE XVIII
Oxygen 408

LECTURE XIX
Animal Oxidations 439

LECTURE XX
Ferments 461

LECTURE XXI
The Functions op the Digestive Organs. 1 484

TABLE OF CONTENTS. xui

LECTURE XXII
The Functions of the Digestive Org,*jvs. II 511

LECTURE XXIII
The Blood. Co.\gulation. Composition 535

LECTURE XXIV
Blood and Lymph , 558

LECTURE XXV
The Elimination of Metabolic Products from the Body 579

LECTURE XXVI
The Relation op the Org.\.ns to One Another 596

LECTURE XXVII
General Metabolism. I 620

LECTURE XXVIII
Gener.\l Metabolism. II 644

LECTURE XXIX
Outlook. 1 663

LECTURE XXX
OULTOOK. II 679

PHYSIOLOGICAL CHEMISTRY.

LECTURE I.
INTRODUCTION.

Physiological chemistry forms an important branch of the large
field of investigation included under physiology. It has, for one thing,
the task of determining the chemical composition of the material from
which the separate tissues in the living organism are formed. A knowl-
edge of the chemical construction of the different organs gives us the
answer to certain questions, and forms the basis for further inquiry.
It is perfectly clear that the organism must receive in its nourishment all
those elements of which its tissues are composed. With the knowledge of
the composition of the separate organs, we obtain, by comparing their
functions, certain interesting views regarding the significance of the dif-
ferent substances which take part in their formation. Closely related to
such problems stands the investigation of metabolism. This has become
almost exclusively the domain of the physiological chemist. We desire,
first of all, to know as much as possible concerning the nutriment that the
organism receives, and especially as regards its utilization in metabolism.
We obtain an insight into such processes by carefully studying the excre-
tions from the organism. The ph}-siological chemist has long since passed
beyond these boundaries. His foremost task is now to ascertain what
becomes of each separate group of food-stuffs in the organism, in what
way it reaches the tissues, and how the cells utilize the different substances
in their metabolism and are built up by them. For a long time we have
not been content with merely contrasting the income with the outgo of
the organism. A final goal of physiological-chemical research will be
attained, when we are able to follow, in every separate phase, each and
everj^ food-stuff from the time of its introduction into the alimentary
canal throughout its entire stay in the tissues until it is finall.y eliminated;
80 that a whole chain, without any missing links, of all the different trans-
formations and complicated processes will lie exposed to our view. We
are still very far from the solution of this problem. To be sure, in recent
years certain progress has been made in our knowledge of metabolism, and
indeed here and there the advance of pure chemistry has placed certain

1

2 LECTURE I.

interesting biological discoveries upon a firm foundation; yet, neverthe-
less, the investigations of the physiological chemist are obliged to halt for
the present before the individual cell. Here for a time is our boundary,
but that it will not prove insurmountable is shown by recent investigations.

Physiological chemistry seeks to accomplish still another result. We
desire to know not merely the manner in which each separate substance
passes through the body, and how it is broken down, but we also wish to
know its relations to the other compounds which are likewise introduced
into the body. This is true especially for our organic food-stuffs. We
wish to know whether they can mutually replace and supplement one
another, and whether it is possible for a representative of one class of
foods to exercise a function usually assumed by another.

Beyond these limits, physiological chemistry sets itself a number of
other tasks which are now only just beginning to be attacked. As little
as we are content with the discovery of the anatomical structure of a
definite organism, but seek to understand clearly its ontogenesis and
phylogenesis, just so little should we be satisfied to trace in the case of a
single individual, or in only one species, all of the processes which may
be referred back to chemical decompositions. Comparative physiological-
chemical research is called upon to explain clearly certain important
processes which now appear enigmatical to us. On the other hand, com-
parative chemical investigation concerning the nature of the bodies and
the metabolism of individuals belonging to different animal species, will
give a new support for purelj' morphological research. The gradual
evolution in the entire animal kingdom takes place step by step parallel
to an ever more delicate and ever more marked specialization of the single
organs. In many cases where the histology of an organ permits some
doubt to arise as to where it belongs, the determination of its function
often gives a clear decision.

More and more the investigation of physiological-chemical processes in
the animal organism extends beyond its more narrow field. For a long
time it has been evident that there is no sharp boundary between the
animal and vegetable kingdoms. More and more it becomes recognized
that the line once drawn was an entirely artificial one. We now know
that countless processes take place in plant and animal organisms which
are common to both. The more this uniformity has found expression,
the more sharply defined have become the differences in the metabolism
of the different members of each kingdom. Everywhere there are tran-
sition stages, and nowhere do we meet with sudden changes. To-day there
is no longer any doubt but that a complete understanding of the pro-
cesses transpiring within the animal organism is only possible when atten-
tion is paid as well to those changes taking place in plant organisms. We
desire to know, moreover, the source of each article of food, how it has been

INTRODUCTION. 3

formed, and to what extent the animal cell is able to build it up for itself.
In such problems as these we meet with deep-seated differences in the
chemism of plant and animal cells.

Physiology and physiological chemistry were at one time a single field
of investigation. The latter, owing to the remarkable progress of the
exact sciences chemistry and physics, has now developed to such an impor-
tant branch of natural science that it is altogether impossible to-day for
any one scientist to master thoroughly in all its details the whole field
of physiology. Little by little, there has developed a sharp distinction
between pure physiology and physiological chemistry. It is clear that
an artificial separation of two such closely related fields of investiga-
tion must work to disadvantage for the development of each, so that it is
fortunate to find it recognized more and more that the whole science of
physiology can only develop satisfactorily when there is an intimate
exchange of ideas between investigators in the two fields. The chemical
decompositions in the tissues do not take place beside the " physiological
processes," but are bound up with them. In no single case can we carry
out any distinction in such a sense; and with those organs in which the
relations between the functions and the metabolism are not known, this is
chiefly because of our limited knowledge concerning the latter. In this
connection we need only recall the whole nervous system.

Finally we must mention the fact that physiological chemistry is con-
stantly approaching new fields of investigation which a few years ago
apparently had almost no connection with it. We have in mind here the
great field of infectious diseases and the changes in the metabolism of the
cells which they occasion. The apparently great gap existing between
the methods of protecting the organism against the substances produced
by micro-organisms and the products given off by the cells under normal
conditions is at last bridged over by means of certain analogies and numer-
ous transitions. Pathology also, in a broad sense, seeks to unite itself
more and more with the field of physiological chemistry. Many a path-
ological process has afforded us a desired physiological e.xperiment, and
conversely it is possible to obtain b\- comparing physiological and path-
ological processes a clearer conception of the latter. It becomes more and
more evident that pathological changes in the tissues and cells should not
be judged entirely from a morphological standpoint. We can easily under-
stand that a disturbance in the metabolism of the cell, if continued long
enough, can make itself evident in the outer appearance. On the other
hand, it is perfectly conceivable that a definite functional disorder may
exist without the anatomist being able to localize it by the means at his
disposal. In no case does it follow necessarily that the limit of discern-
ible degeneration is a measure for any given functional disturbance.
Again, an abnormal morphological change may cause but a slight dis-

4 LECTURE I.

turbance in the metabolism of tlie cell. At present we are very far from
gaining insight into the physiological processes concerned in cell-metab-
olism, and still less should we expect to obtain already a clear picture of
pathological disturbances. This opens up for us a particularly enticing
field for further investigation, the foundations of which must rest upon
physiological chemistry. It is apparent from this cursory glance that the
tasks of physiological chemistry are extremely varied.

In order to be able to form a correct judgment of results brought for-
ward in a field of investigation, it is necessary to decide in the first place
whether the methods employed are satisfactory and rest upon a firm
basis. The methods used by the physiological chemist are for the most
part those of pure chemistry. Such experience forms the basis of his
work. We shall soon see that in spite of the fact that in many cases
perfectly similar methods are employed, there is a marked difference in the
two fields of investigation. Chemistry, we know, is an exact science.
This designation means, more than anything else, that the chemist arrives
at his conclusion by carrying out from case to case a direct proof, in a
perfectly objective manner. If he discovers a new substance, he is able
by various processes to so purify it that by its crystalline appearance, the
results of analysis, its melting-point, molecular weight, etc., and by a study
of the substances he can form from it, he can decide whether it is a simple
substance or a mixture. Finally, he can decompose the substance, it may
be by hydrolysis, or perhaps by oxidation, or by reduction, thus transform-
ing it into constituents of known composition, or into some other well-
known compound, and in this way eventually establish its composition in
all its details. But even after all this work the chemist is not satisfied.
He is not absolutely positive that he has a substance of definite constitu-
tion until he has succeeded in effecting its synthesis. To be sure, meta-
physical speculations also play an important part in the domain of pure
chemistry, and rightly. This has long since been shown to be justifiable
on account of the fruitfulness of such speculations. The chemist must
never forget, however, where the facts end and where the hypothesis
begins.

Let us see now how the physiological chemist conducts his proofs. One
might be tempted to believe that an insight into the chemical processes
which take place in the organism would be obtained soonest if we chose for
our study the simplest form of organized being, the single cell, i.e., a unicell-
ular individual. A little thought, however, shows us that the morphological
unity of the cell corresponds to an uncommonly complicated cell-mechan-
ism. Each cell-unit here takes up nutriment, decomposes and assimilates
it, and eventually breaks it down to deposit at last the final products of
metabolism. All of these processes take place in a single minute cell.
From this point of view, every morphological differentiation must in a

INTRODUCTION. 5

certain sense be regarded as a simplification of the functional processes.
The more localized the .separate functions are, and the more they pertain
to certain organs onl\-, the greater becomes the possibilitj- of our succeed-
ing in studying them by themselves and explaining them. In this sense
the most suitable objects for our study are those highlj' complicated beings,
the vertebrates.

In order to show the value of the results obtained by physiological-
chemical investigation, a few examples may be cited briefly. We will
disregard here that part of the field which concerns itself with the knowl-
edge of the separate components of our food and the substances which go
to form our tissues. In such cases it is evident that the physiological
chemist will make use of the same methods for determining the value of
his work as does the pure chemist. He will, furthermore, proceed in pre-
cisely the same way as we have described for a chemical investigation, and
will thus establish the proof for the constitution of a definite compound.
Here physiological chemistry is constantly receiving much aid from the
field of pure chemistry, and in fact this part of the work has been
developed by trained chemists.

We will choose here, as an example, a question which has been asked
repeatedly, namely this: What becomes of a definite compound after it
is introduced into the animal organism, in what way is it broken down,
and in what form is it finally excreted? We will use for our illustration
a very important discovery, rich in results, which we owe to Wohler.
This scientist was interested to learn what became of benzoic acid after it
was introduced into the intestine. He was unable to detect it in the
urine, nor could he find there any substance which he recognized as
one of its lower derivatives. On the other hand, he did find in the urine
another acid, hippuric acid, which is closely related to benzoic acid. This
is formed, as we shall see later on, by the combination of benzoic acid with
glycocoll, the latter substance being formed by the hydrolysis of albumin.
Hippuric acid on being boiled with strong mineral acids or alkalies is
decomposed into these two constituents. Wohler, by establishing the
fact that the benzoic acid which he introduced into the organism left it in
the form of hippuric acid, proved for the first time that .syntheses may take
place in animal organisms. In this way the path was broken and the ob-
struction to the development of physiological chemistry which had existed
for years was removed. Up to that time it was regarded as an established
fact that onh' plant cells were capable of accomplishing synthetical work,
while those of the animal organism could only effect decomposition.
This first observation of Wohler was particularly fruitful, and inspired
a great deal of similar work, so that to-day we are perfectly justified in
ascribing complicated syntheses to the action of the animal cells. If we
attempt to subject Wohler's method of proof to close critical analysis, we

6 LECTURE I.

find, first of all, that it is indirect in character. Here we meet with the most
remarkable point in all physiological-chemical investigation. A very
large part of it is based upon indirect proofs. Its inadequacy is clearly
shown if we choose for illustration, instead of the above very transparent
example, something more complicated, such as, for example, the question
now in the foreground of general interest with regard to the formation of
sugar from other sources than carbohydrates. By extirpating the pan-
creatic gland of a dog, its carbohydrate metabolism may be so disturbed
that sugar is constantly eliminated in the urine, and one would naturally
think a priori that in such a case it would be possible to determine with-
out difficulty whether, for example, albumin or fat can cause secretion of
sugar in the urine. As a matter of fact, this secretion of sugar continues
even after all carbohydrates are completely eliminated from the nourish-
ment. We may assume this to prove that albumin and fat can cause
secretion of sugar in the urine. Although we are justified in drawing such
a conclusion, it is not necessarily a correct one. A result from a given
experiment can lead to different conclusions according to the standpoint
assumed by the individual investigator. In this case it is possible to
explain the continued secretion of sugar in another way. The animal
organism possesses constant reserves. Their extent has only recently
been realized. From them, and especially from carbohydrate stores, the
sugar may have its source. The conclusion that the animal cell is capa-
ble of forming sugar from other sources than the carbohydrates can only
be drawn with certainty after it has been established that the organism
has no more carbohydrates at its disposal. Not till this has been clearly
shown will the above conclusion rest upon a firm basis. It remains still
undecided, even if the carbohydrates as sugar-formers are fortunately
excluded, as to whether fats and albumins belong to this class of com-
pounds. Now it has been often observed that in feeding albumin to a dog
with no pancreas the elimination of nitrogen runs practically parallel
to that of sugar. This repeatedly established relation between the break-
ing down of albumin and the formation of sugar has been given as a direct
proof for the formation of sugar from albumin, and in fact one might
be tempted to assume that this is actually a direct demonstration.
E. Pfltiger, whom we have to thank for a detailed critical review of all the
work in this field, is of an altogether different opinion. If we assume as
correct that the elimination of sugar and of nitrogen increases at an equal
rate, we are still far from being justified in assuming that the increase in
sugar is directly due to the breaking down of the albumin which is taking
place. The cells of a dog with no pancreas, and those of a diabetic, have
not lost entirely the power of consuming sugar, and in all cases a part of
the sugar formed is burned up. If now albumin be fed, it will also be
burned; in other words, there will be set free in the tissues of the organism

INTRODUCTION. 7

a definite number of heat units which it can use in performing its func-
tions. By means of the calories of heat coming from the albumin the
organism is spared a corresponding amount which were otherwise taken
from non-nitrogenous material. We may assume that the cells, which
moreover are capable of consuming sugar only with great difficulty, now
do not use so much of it. Jlore and more unchanged sugar circulates in
the tissues and in the blood, and since the kidneys, as we shall see later,
are sensitive to the slightest increa.se of sugar in the blood over the normal
and serve to remove all such excess, it follows that there must necessarily
be an increase in the elimination of sugar. According to this hypothesis
the action of albumin is an indirect one as regards the sugar. Naturally
this explanation is not necessarily the correct one. We have mentioned
these experiments and the two explanations of the results obtained briefly
in order to show by a somewhat complicated example how varied the con-
clusions may be that are drawn with regard to an apparently simple prob-
lem. It would not be difficult to cite numerous other examples to illustrate
this point. Later on we shall repeatedly come back to these indirect proofs
and mention again and again the fact that it is of fundamental importance
for the further development of all physiological-chemical investigation
that it should always be clearly and sharply recognized as to what extent
we are justified in speaking of jads, and at what place the indirect con-
clusions, corresponding to the still unsettled part of our field of investiga-
tion, begin. When such a gap is discovered, it is our duty not to r&st satisfied
until all of the conclusions have been subjected here also to direct proof.

Before taking up the discussion of ways and means to accomplish this
end, we will turn back once more to the synthesis of hippuric acid in the
animal organism. This was established indirectly, and its assumption
rests solely upon probability. After introducing benzoic acid into the
organism of a mammal we find a corresponding increase in the amount of-
hippuric acid in the urine. It is a fact that hippuric acid can be formed
from benzoic acid and glycocoU. The chemist is able to make hippuric
acid in the laboratory from these two components, but under conditions
which it is impossible to realize in our tissues. It requires a high tempera-
ture, considerable pressure, and the exclusion of water. We have, how-
ever, long since been forced to the conclusion that the cells have the power
of causing chemical reactions to take place which require entirely dif-
ferent conditions when carried out in a test tube. We are satisfied if an
observed chemical process does not outwardly contradict our general
experience. We base our explanations of the chemical decompositions
taking place in the animal organism upon the results of chemical research,
and seek to go farther and bridge over all the large gaps which we meet
with everywhere on account of our insufficient knowledge of metabolic
processes. Here also we must be conscious that we are only speaking of

8 LECTURE I.

probabilities, and in no case should it be credited as if resting upon facts
established experimentally.- Analogies in many cases are without doubt
very valuable, and often form the skeleton upon v^^hich we can build further.

We receive a new impulse and gain a new point of view with every
advance made by pure chemistry concerning substances of physiological
interest. Our task is to utilize each discovery thus made and to give it a
strictly objective test with regard to its application to the processes taking
place in the tissues. Here again we meet all too frequently with hypotheses
which are stated as facts. We hardly need to mention how extraordinarily
restraining the direct amalgamation of these entirely different elements is
for a healthy progress in the knowledge of chemical processes in the animal
organism. For these reasons Woiiler's experiment proves positively
merely that when benzoic acid is introduced into the system it causes an
increased elimination of hippuric acid. It must remain an open question
as to whether the benzoic acid introduced stands in direct relation to the
other acid or merely indirectly causes its formation. In this particular
case, however, the latter case seems scarcely probable, although we must
make such a limitation unless we propose to draw our conclusions beyond
the realms of fact.

We must now mention an important aid which the chemist makes
use of constantly in his experiments, which are often indirect in nature.
We refer to the control experiment. It is clear that there is nothing
to be gained by merely feeding an animal with benzoic acid and deter-
mining subsequently the amount of hippuric acid eliminated. We must
first learn how much hippuric acid the animal in question eliminates under
normal conditions. If the experiment is to be made convincing, the amount
of hippuric acid contained in the urine of one and the same animal fed
uniformly must first be determined and this continued for several days.
Then for a time a little benzoic acid should be added to the food, which
otherwise must remain qualitatively and quantitatively the same as before,
and again the hippuric acid be determined in the urine. If now the experi-
ment be continued for another period of several days in which no benzoic
acid is fed to the animal, then, if the whole experiment is consistent, it
will be possible to determine whether the benzoic acid stands in any
relation to the elimination of hippuric acid.

The uncertainty of the significance of experiments made with animals
is in many cases greatly increased by tlTe fact that individual variations
often play an important part. Many contradictions to be found in the
literature are due solely to the fact that the experiments were not carried
out long enough. We must not only require that such experiments should
be carried out in a single individual for quite a length of time, but in dif-
ferent individuals of the same animal species as well. It is, furthermore,
of great value to make experiments with different species of animals, for

INTRODUCTION. 9

frequently they behave altogether differently physiologically. It is
apparent already from these brief remarks what great demands are laid
upon the experimentation of the physiological chemist. He always has to
deal with complicated processes. He is acquainted usually only with the
initial and the final products of the metabolism, and is compelled to clear
up theoretically the whole chain of transformations which are necessary
for the formation of the latter from the former. Here and there it is
possible to get hold of intermediate steps, and thus we encroach more and
more upon the great domain of the unknown.

A considerable advance in the subject was made when experimentation
was begun upon surviving organs rather than upon the whole organism.
Here the initial and end products are more closely related, or at least
apparently so, although here also, as soon as the change in cell substance
begins really to take place, the complication is naturally practically as
great as in following one substance through the whole body. Experimen-
tation with surviving organs has in itself quite a number of advantages.
In many cases we are able to change an indirect proof into a direct one.
We are able to work out accurately the composition of a definite organ.
We can definitely decide the question as to whether it has stored up in it
sufficient amounts of definite substances to cause the formation of certain
compounds, and thus determine positively whether the organ makes use of
a substance introduced into it in a definite process.

Let us return again to our hippuric acid hypothesis. It is possible to
establish which organ is capable of carrying it out. G. Bunge and O.
Schmiedeberg have shown that the kitlneys of mammals, or more accu-
rately those of a dog, are capable of forming hippuric acid from glycocoll
and benzoic acid. They caused a dog to bleed to death, cut out its kidneys,
and introduced defibrinated blood through the arteries of the kidneys and
allowed it to flow out through the renal veins. On introducing glycocoll
and benzoic acid, there appeared hippuric acid in the blood and in the
liquid emptying out through the ureter. A control experiment with the
second kidney showed that it as well as the blood from the first kidney was
free from hippuric acid. If benzoic acid but no glycocoll was introduced
into the blood, the amount of hippuric acid formed was extremely small.
The conclusion to be drawn from this experiment is that benzoic acid
effects the formation of hippuric acid because it is itself used in
tjie synthesis. However, this fact is not yet alasolutely proved. The
objection may still be raised that hippuric acid may arise from another
source. The formation of this acid is, at best, a very complicated process.
We have, on the one hand, the kidney containing a very complex tissue,
and, on the other hand, the blood with its constituents. As a matter of
fact, it was not possible to effect the above synthesis after the red
corpuscles were removed from the blood.

10 LECTURE I.

Another significant advance in the i<nowledge of chemical processes which
take place in the animal organism was caused by the discovery that it was
possible to work out certain processes by means of extracts of tissue. We
are furthermore fortunately able in many cases to isolate the active prin-
ciple. The great advantage of such experiments is clear from the fact that
an extremely small amount of these products, called ferments, is capable of
causing considerable change without itself appearing in the end products of
the reaction. It has been attempted to effect the hippuric acid synthesis in
this way by means of a ferment which has been isolated from the kidneys.
This has not yet been done satisfactorily. An instructive example of
great significance as regards the use of such tissue extracts and of the
ferments obtained from them is found in the recent experiments to form
uric acid from the purine bases, investigations made by Horbaczewski,
Wiener, Spitzer, Schittenhelm, and Burian. The first-mentioned has
shown that purine bases added in the presence of oxygen to an animal organ
which has been macerated to a paste causes an increase of uric acid. Against
this experiment the objection may be raised that it is not conclusive. The
pa.ste itself contains some purine bodies and perhaps substances of unknown
nature which stand in close relation to uric acid. The purine bases added
may in some way have an indirect action upon the given synthesis. The
proof becomes much more satisfactory if instead of using the whole organ,
we make use of a ferment extracted from it. To be sure, the nature of this
ferment is not known, but we know its action. We can free it completely
from purine substances, and furthermore we require but a small amount of
it. It is of great significance with regard to our conclusions, that it is pos-
sible here to follow the experiment quantitatively. We can weigh accu-
rately the amount of purine bases added, and similarly the amount of uric
acid formed, so that we are now able to establish sharply the relation
between the purine bases and the uric acid. This method has still further
advantages. It has been possible to identify certain intermediate products
formed in the transformation of certain purine bases into uric acid, and to
establish the fact that certain organs possess ferments which are capable of
breaking down the uric acid formed. In this way it is at once possible to
establish clearly the complete metabolism of purine substances. It would,
of course, be unsafe to apply the results of such experiments without further
investigation to the processes taking place in the living organism. It is
perfectly conceivable that the conditions prevailing in the tissues may be
entirely different from those prevailing in the fermentation experiments
carried out artificially. Such a limitation holds for all investigations
carried out with ferments, and especially those with digestive ferments. In
such experiments the ferments develop their action under entirely changed
conditions. We can merely imitate the temperature; further than this we
are practically helpless. In the alimentary canal, for example, absorption

INTRODUCTION. 11

takes place immediately hand in hand with the hydrolysis of food by the
ferments of the digestive juices. The decomposition products are imme-
diately taken away. We are still entirely ignorant of the manner in which
each individual ferment does its work, how the different ferments assist
one another, and how their work is influenced by other factors. At all
events, it is evident that the decomposition of food takes place much more
rapidly than in a test tube. All the products of the decomposition remain
in the latter case, and serve to hinder the further action of the ferment, or
perhaps even cause it to act in a different direction. On the other hand, in
the test tube we are often able to identify products which otherwise escape
our observation owing to the rapid absorption in the bowels. It is possible
here also to draw conclusions only by combining experiments; that is, on
the one hand we will study digestion as accurately as possible in the test
tube, following it up in its separate phases, and, on the other hand, we must
attempt to identify the products of digestion in the bowels themselves; in
this way we gradually draw a picture of the entire process of digestion.
In an entirely similar manner the metabolism of purine substances must
be studied in the whole organism in order to find out how far the facts
thus ascertained agree with the results of experiments with ferments.

We must consider still another important condition, namely, the concept
of quantit}-. In physiological-chemical experiments this is too frequently
neglected. Its importance is perfectly obvious. We must always require
that every chemical process taking place in the organism be followed
quantitatively. Qualitative experiments are prone to lead to great errors,
and it is never possible to recognize clearly by means of them the relation
between individual products. We must always know how much of thi.s
or that substance has been changed over into a definite product. Often-
times a minor process will otherwise be considered the essential one simply
because it was easy of discovery, whereas the main change may be entirely
overlooked.

It is almost superfluous to mention the fact that the methods employed
must be suitalile for the prolilem to be investigated. Every investigation in
the field of physiological chemistry must start out with a critical examina-
tion of the value of available methods. We must clearly recognize the
sources of error and take them into consideration, especially when definite
conclusions are to be drawn from any discovery. The methods are the
foundation pillars in every experimental investigation. Every advance is
closely dependent upon them, so that we must lay great stress upon their
final development. The great importance in the improvement of methods
too often falls into the background, especially in physiological-chemical
investigation, and apparently more weight is laid upon the more or less
fruitful hypotheses. It must not be forgotten, however, that essentially
new facts are usually closely connected with the discovery of new methods.

12 LECTURE I.

The latter alone cause the science to progress upon a solid foundation.
They assure an objective investigation, and above ail else one that is
free from prejudgment. Certainly hypotheses and speculations are of great
value, and their importance should not be underestimated. They form
the framework upon which we can build further. The facts, however,
should never be adjusted in accordance with them. The facts, and never
the hypotheses must always be decisive. This warning is not unnecessary,
for in contrast to the exact sciences such as chemistry and physics,
here in physiological-chemical investigation the hypotheses step boldly
into the foreground, especially in questions concerning metabolism, and
in particular that of the cell substance.

We make these few preliminary remarks in order to show at the start
the nature of our lectures and the principles which are authoritative. It
will be our aim to define as sharply as possible what discoveries are to be
regarded as well-established facts and at what place the probability
proofs are justifiable. Above all else we shall strive to follow every
separate food-stuff from its introduction into the organism to its complete
breaking down and the elimination of the end-products in order thus to
obtain a comprehensive view of its behavior in the organism and
its participation in metabolism. We shall intentionally consider the
building materials and composition of the separate organs only in
special cases. This knowledge we acquire in studying metabolism.
A consideration of the quantitative relations in which the different sub-
stances are present would be of use to us only when the separate values
are based upon a broad foundation and upon a great many observations.
For the present our methods are not adequate to give us a satisfactory
picture of the building up of the separate tissues. Neither is our
knowledge sufficient to permit the valuation of the results for compara-
tive studies, nor are we in general in a position to draw conclusions
with regard to the functions of certain organs from a knowledge of
their composition. In the special cases where this is possible we shall
speak of it.

LECTURE II.
CARBOHYDRATES.'

In General — Monosaccharides — Glucosamine — Glucuronic
Acid.

The carbohj'dratp.s are extremeh- abundant in nature. They take a
prominent share in the building up of the vegetable kingdom, and play an
important part as food in animal economy; while on the other hand, com-
pared with the protein bodies, they scarcely come into consideration at all
as building materials for the animal tissues and cells. The most important
representatives of this class of bodies have been known for a long time,
especially cane sugar, which before the beginning of the Christian era was
obtained in India in a solid form by boiling down the juice of the sugar-
cane. To-day, besides the sugar-cane, the sugar-beet ' forms an important
raw material. Again, grape-sugar has been known for a long time, and
was first discovered in honey although prepared pure for the first time
b}' Marggraf in the middle of the eighteenth century. In the year 1615
Bartolleti ^ isolated a third member of this group from milk, namely
milk-sugar. If we add to these cellulose and starch we have named
all of the members of the carbohydrate group which were known up to
the time that the study of organic chemistry as we know it to-day began
as a result of the experiments of Lavoisier and Scheele. If we disregard
a few isolated although very important observations — e.g., Kirch-
hoff's discovery that starch was changed into grape-sugar by boiling with
dilute acids,' and that the same process could be brought about by a sub-
stance found in grain or malt ' — very little was known in chemistry,
and consequently in physiology, concerning carbohydrates up to within
very recent times, and this period of darkness disappeared only with the
important investigations of Kiliani and of Emil Fischer especially.

' The following references cover this field: — Emil Fischer: Ber. 23, 2114 (ISgO).
E. O. V. Lippmann: Die Chemie der Zuckerarten (1904). B. Tollens: Kurzes
Handbuch der Kohlehydrate (1S9S).

' Discovered by Marsgraf (1747); Ber. Berlin. .\kad. Wissensch. 79, 1749.

' EncycIop:cdia dogmatica, 101 j.

« J. d. Pharm. 74, 199 (ISll).

' Schweigger's J. 14, 3S9 (1814).

13

14 LECTURE II.

In the course of the following discussion we shall see how closely the
development of the chemistry of carbohydrates follows the general develop-
ment of chemistry, and especially that of stereochemistry and the theory
of structure, and what a comprehensive outlook dawned all at once
for the whole field of biology.

The carbohydrates are all composed of the elements carbon, hydrogen,
and oxygen, and these are the same elements that are found in fats. The
two classes of compounds, however, contain these elements in different
relative amounts. Oxygen and hydrogen in the former are present in the
ratio 1 : 2, which is the same as in water. This is the reason that the
name carbohydrates has been given to the group. Many other compounds
which do not belong to the sugar group, for example acetic and lactic
acids, are, however, also composed of the same elements and in the same
ratio. Formerly, the carbohydrates were defined as containing six, or a
multiple of six, carbon atoms. This limitation was shown to be incorrect
by the discovery of sugars containing less than six atoms of carbon, and by
the synthesis of sugars with seven, eight, and nine carbon atoms. It is in
fact impossible to give a sharply-defined, satisfactory definition of a carbo-
hydrate, for to some extent the individual members of the group have very
different properties from one another. In general, the carbohijdrates are
aldehyde or ketone derivatives of pohjatomic alcohols.

As is the case with almost all branches of physiological chemistry, so
here, as has already been indicated, it was only possible to obtain a clear
idea of the formation and transformations of carbohydrates in the animal
and vegetable organisms after the compounds in question had been pre-
pared synthetically. It was Emil Fischer who first succeeded in this
effort, by preparing from glycerol — the same glycerol which we shall
meet with again in the discussion of fats — by gentle oxidation, a sub-
stance with the typical properties of a sugar. This compound, called
glycerose, contains, to be sure, only half as much carbon as grape-sugar. As
it was found possil)le to prepare by the action of dilute alkali upon two
molecules of glycerose a true sugar with six atoms of carbon, there was
no longer any doubt that glycerose was to be regarded as a member of
the carbohydrate group. This synthesis is of especial value to us, as it
establishes a relation between the fats and the sugars. Finall_v, it was
even possible to effect the synthesis from the elements; for starting with
formaldehyde, CHoO, Emil Fischer succeeded by polymerization
(6 X CH2O = C6H12O6) in obtaining the same sugar as that made from
the glycerose prepared from glycerol.'

This complete synthesis isparticularly interesting to us, because some time

' Ber. 23, 2114 (1S90); Die Chemie der Kohlehydrate und ihre Bedeutung fiir die
Physiologie, Berlin, 1894; Synthesen in der Purin-und Zuckergruppe, Braunschweig,
1903.

CARBOHYDRATES. 15

before this Adolf v. Baeyer ' had explained in exactly the same way the
formation of carbohydrates in plants. According to this conception, which
up to the present time has not been proved absolutely, the leaves contain-
ing chlorophj-U reduce the carbon dioxide of the air to formaldehyde, and
the latter is transformed into sugar by condensation. The formation of
sugars containing a different number of carbon atoms can be similarly
explained with the help of the same hypothesis. It is, indeed, perfectly
possible that the building up of the higher sugars by nature takes place
through the same intermediate stages as have been observed in the arti-
ficial synthesis.

Now, an accurate examination showed that the sugar obtained from
glycerose, or from formaldehyde, containing si.x atoms of carbon was not
identical in all its properties with grape-sugar. It was, therefore, given
a special name, acrose. Biot ' made the important discovery that cane-
sugar rotates the plane of polarized light. This property, which other
sugars found in nature likewise show, was quickly utilized technically
for the quantitative determination of cane-sugar in cane-juice, etc' It
proved to be also of considerable aid in distinguishing the different
kinds of sugar from one another. Now acrose does not have this property:
it does not rotate the plane of polarized light. The reason for this is that
acrose is composed of components each having the opposite effect upon
polarized light, and as a matter of fact it is possilile to decompose acrose
into these unlike individuals. According to the conditions of the experi-
ment, it may be changed into fruit-sugar or mannose or grape-sugar.
Herewith the final step in the artificial synthesis of sugars such as occur
in nature was accomplished.

The optical activiti/ of almost all natural products — a property which for
a long time served to distinguish natural products sharply from artificial
ones, and gave support to the theory that a special force peculiar to a living
organism was necessary for the production of such compounds, until at last
here also successful synthetical chemistry made a breach in the wall which
had been considered as impregnable — was first explained by the well-known
fruitful hypothesis of Le Bel and van 't Hoff ^ (1874). These two scien-
tists independently traced the asymmetrii of the molecule, which Pasteur ^

' Ber. 3, 63 (1S70).

' Compt. rend. 10, 264; 16, 619 (1843).

' Clerget: Compt. rend. 16, 1000 (1843); 22, 1138 (1846); 23, 256 (1846); 26, 240
(1848).

' Cf. van't Hoff: Die Lagerung der Atorae im Raume. Dix ann^e dans I'lii-stoire
d'une tWorie. La chimie dans I'espace (1875). K. Auwers: Die Entwicklung der
Stereochemie (1890).

' Leijons de chimie profess^es en 1860. Paris, 1861. See also H. Landolt: Das optische
Drehung.svermogen organischer Substanzen und dessen praktische .\nwendungen,
Braunschweig, 1898. A. Werner: Lehrbuch der Stereochemie, Jena, 1904.

16 LECTURE II.

had ingeniously brought forward in order to explain the optical difference
between dextro-tartaric and laevo-tartaric acids, to the individual carbon
atom. This atom is in combination with four different masses. Every
asymmetric carbon atom in a compound causes the possibility of two
optical isomers, one rotating the plane of polarized light to the right, and
the other to the left. We can illustrate this best, according to van 't Hoff,
by imagining the valences or affinities of the carbon atom extending to-
wards the apexes of a tetrahedron in the center of which the carbon atom
itself is placed.

Fig. 1.

(Ri, R2, R3, R4, are the four different masses with which the carbon
atom is combined.)

The above drawing represents this kind of isomerism. The two forms
are in the same relation to one another as an object and its reflected
image, or as a right and left glove; i.e., they cannot be superposed one upon
the other, so that the corresponding parts will all coincide. There are,
then, three possiljle mocUfications in the case of every carbon compound
containing an asymmetric carbon atom, namely, two optically active forms,
and one which is inactive, being composed of an equal number of molecules
of each of the other two forms. In the last case the two asymmetric
carbons, although both active, have an equal and exactly opposite effect
upon polarized light, so that they neutralize one another.

If these assumptions are correct, then if there are two or more asymmet-
ric carbon atoms in the molecule, the number of possible optical isomers
must increase regularly and amounts to 2" where n is the number of asym-
metric carbon atoms. This theory has been confirmed empirically
to a most remarkable degree, and, indeed, in no part of chemistry has
the work of Le Bel and van 't Hoff been so strongly supported as in the
development of carbohydrate chemistry according to this point of view
by Emil Fischer.

In advance, it may be mentioned, for example, that there are several
different sugars having the empirical formula C6H12O6. Of these we need
mention only d-glucose, mannose, and galactose. Now all of these sugars
contain, as shown by the following general structural formula, no less than
four asymmeti'ic carbon atoms:

CARBOHYDRATES. 17

COH

I
*CH.OH

I
*CH.OH

I
*CH.OH

I
*CH.OH

I
CHo.OH

According to the rule given above, 2* = 16 different compounds should
exist having such a structure. There is really no doubt concerning this,
for already no less than twelve isomers representing six optical pairs have
been isolated. For each one of these the geometric constitution is ex-
plained by the theory, and the configuration of each single molecule
represented by a definite structural formula. The following is a summary
of the configuration formute given by Fischer to the hexoses,' in which
the asvmmetric carbon atoms are marked with an asterisk.

COH

I
H— *0H

I
H— *— OH

I
HO— ^:=— H

I
HO— *— H

CH2.OH

^-Mannose

COH

HO— *— H

1
H— *0H

HO— *— H

H— *— OH

Ah.

,0H

Mdose

COH

I
HO— *H

HO— *— H

I
H— =^— OH

i
H— =^=— OH

I
CH3OH

<f-Mannose

COH

I
H— -— OH

HO*— H

H— *0H

HO— *— H

I
CHoOH

f/-Idose

COH

I
HO— *— H

I
H— *— OH

HO— *— H

I
HO— =^— H

I
CH2OH

^-Glucose

COH

I
H— =:=— OH

1
H— *— OH

I
HO— *— H

H— *— OH

I
CH2OH

Z-Gulose

COH

I
H— *— OH

HO— *— H

H— *— OH

I
H— -— OH

1
CH2OH

d-Glucose

COH

HO— *— H

I
HO— *— H

I
H— *— OH

I
HO— *— H

CH2OH
rf-Gulose

' The configuration forraulip for sugars with less carbon liave also been worked out.

18

LECTURE

II.

COH

1

COH

1

COH

COH

1

HO— *— H

1

H— *— OH

j

H— *— OH

1

HO— *H

H— *— OH

1

HO— *— H

1

H— *— OH

1

HO— *— H

H— *— OH

1

HO— *— H

j

H— *— OH

HO— *— H

1

HO— *— H

1

H— *— OH

1

HO— *— H

H— *— OH

1

CH2OH

CH2OH

CH2OH

CH2OH

Z-Galactose

d-Galactose

Z-Talose

d-Talose

COH

COH

1

COH

COH

1

HO— *— H

H— *— OH

1

H— *— OH

1

HO— *— H

HO— *— H

H— *— OH

1

HO— *— H

1

H— *— OH

HO— *— H

HO—*—

1

H— *— OH

HO— *— H

H— *— OH

1

H— *— OH

1

HO— *— H

1

H— *— OH

1

CH2OH

CH2OH

CHoOH

CH2OH

Not yet prepared.

The detailed discussion here of these purely chemical problems is com-
pletely justified on account of the great significance which these investiga-
tions have for biology, and as a matter of fact it will not be possible to
understand clearly the metabolism of carbohydrates without having a
thorough knowledge of such structure questions as we have briefly touched
upon.'

The ways and means by which sugars rich in carbon are prepared syn-
thetically from those with fewer carbon atoms are not without interest
for biology. All the compounds represented on the preceding page con-
tain an aldehyde group, and aldehydes are capable of combining with
hydrocyanic (prussic) acid. By saponifying the cyanhydrins and sub-
sequent reduction, a new sugar is obtained, as E. Fisher and later Kiliani ^
showed, which contains more carbon than the original sugar. In this way,
it is possible to prepare not only hexoses from the simplest members of
the carbohydrate group, but sugars with seven, eight, and nine atoms of

' Since we shall meet with the same point of view in the case of other classes of
substances, especially the proteins, the student is advised to refer to some of the books
on the subject.

■•' Ber. 18, 3066 (1885); 19, 221, 767, and 1128 (1886).

CARBOHYDRATES. 19

carbon as well.' There appears really to be no limit to the applicability of
the synthesis.

Perhaps this synthesis throws some light upon the formation of the
different sugars in plant organisms, for it must be often necessary for
them to build up from sugars containing a small number of carbon atoms
those with more carbon. The possibility of such transformations shows
that there is no sharp distinction between the separate sugar groups contain-
ing different numbers of carbon atoms, and unites, both chemically as well as
biologically, the different chisses to a large unit which becomes even more
closely related by reason of the fact that the artificial representatives of
the same group such as grape-sugar, mannose, and fruit-sugar, can be
changed into one another by successive oxidation and reduction.

The large number of sugars prepared synthetically, some of which have
not yet been found in nature, together with the natural sugars are sub-
divided into groups. We distinguish, in the first place, between the more
simple sugars called monosaccharides and compound sugars called poly-
saccharides. The latter may be regarded as formed from two or more
molecules of the former with elimination of water, and, as a matter of
fact, the simpler sugars may be formed from them by hydrolysis.

The monosaccharides again are divided into subclasses governed by the
number of carbon atoms in the molecule. Thus we have a diose (glycol

HCO
aldehyde, or glycolose, | ) which is the simplest possible sugar,

CH2OH
and trioses, tetroses, pentoses, hexoses, heptoses, etc. We have seen, further-
more, that, in general, a sugar is a polyatomic alcohol containing either a
ketone or aldehyde group. Corresponding to this, from the trioses on, we dis-
tinguish between aldo.ses (aldehyde alcohols) and ketoses (ketone alcohols).
Again, an important type which we frequently meet with in nature is that of
the methyl derivatives of these sugars; thus we have fucose (from rockweed,
known botanically as fucus), rhodeose (from jalap) and rhamnose (pre-
pared from numerous plants), which are all designated as meth>/l pentoses.

Of all the numerous sugars, but few are found in the animal organism,
and only a few play any considerable part as forms of nourishment, al-
though we must admit that our present knowledge concerning the phys-
iological significance of numerous sugars found distributed throughout
the vegetable kingdom, partly free and partly combined with other
substances, is still far from being complete. Members of the last-men-
tioned class of substances, the number of which is extremely large are
known as glucosides, and as such we designate all substances which are
more or less readily decomposed into a sugar on the one hand and a com-
pound either aromatic or aliphatic in nature on the other. Such decom-

' Emil Fischer: Ann. 270, G4 (1892).

20 LECTURE II.

positions may be effected by ferments (emulsin, myrosin, betulase, etc.)
as well as by chemical reagents. Thus, for example, amygdalin breaks
down by the action of emulsin into two molecules of grape-sugar, one
molecule of benzaldehyde, and one of hydrocyanic acid:

C20H27NO11 + 2 H2O = 2 CeHisOe + CeHsCHO + HCN.

The structure of these compounds has been cleared up perfectly by Emil
Fischer,' who succeeded in making sugar combine with alcohol and similar
substances by the mere action of dilute hydrochloric P.cid.^ The glucosides
are in fact compounds perfectly analogous to the polysaccharides, both
being formed by the combination of two molecules of simpler compounds
with loss of water, although in the former case the molecules reacting are
unlike, whereas in the case of polysaccharides only sugar molecules are
concerned. Not only monosaccharides, but polysaccharides take part
in the formation of true glucosides, as Emil Fischer showed in the case
of amygdalin.^ Such observations are of great value for biology, as they
permit us to consider the formation of such large classes of substances
from a single point of view.

The significance of the glucosides in the narrower sense of the economy
of the animal organism has up to the present time been but slightly inves-
tigated. Without doubt a part of the sugar contained in the organism is
in such a form, and perhaps this enables such sugar to escape combustion.
It is only recently that such substances have been carefully studied. The
investigations of Thierfelder ■* upon cerebron, a substance isolated by
means of indifferent solvents from the human brain, may be mentioned in
this connection. On being subjected to hydrolysis this substance took up
two molecules of water and formed one molecule of cerebronic acid, one of
sphingosine and one of galactose:

C48H93N09 + 2 HoO = C25H50O3 + C17H35NO2 + CfiHiaOe.

A similar glucoside is the glycoproteid prepared by Schulz and Ditthorn '
from the albuminous glands of the frog, w-hich on being subjected to
hydrolysis yields among other products an amido-sugar, galactosamine, a
result which is closely analogous to the finding of glucosamine by Fried-
rich Mtiller " in the mucin substances of the human respiratory organs.

' Ber. 26, 2400 (1893).

= For example: C,H,,Oe + CH3OH = C„H„0, . CH3 + H,0.

' Emil Fischer: Ber. 28, 1508 (1895). From amygdalin the yeast-enzyme splits off
one molecule of sugar, and forms a new glucoside called mandelonitrile-glucoside,
which emulsin decomposes completely into sugar, benzaldehyde, and hydrocyanic acid.

* Z. physiol. Ch. 43, 21 (1904) and 44, 366 (1905).

"■ Ibid., 29, 373 (1900); 32, 428 (1901).

" Sitzungsber. Gesellsch. Forderung gesamt. Naturwissensch. zu Marburg. 1896i
6; 1898, 6.

CARBOHYDRATES. 21

Later on, when we come to consider the proteins, we shall have to take up
these substances in detail.

From the group of glucosides, furthermore, there are derived a great
many compounds, some of which have strong toxic properties and are very
important drugs, their pharmaceutical value having been discovered purely
empirically.' Of this large group of such compounds we shall mention
only the saponin substances, phloridzin, salicin, helleborin, and the digitalis
glucosides (digitalin, digitonin, digitoxin)^ Finally, it may be stated
that alizarin, the well-known red dye, likewise occurs in nature as a gluco-
side (ruberythric acid) in madder root {Rubia tinctorum). This gluco-
side has lost most of its practical importance on account of the famous
synthesis of alizarin by Graebe and Liebermann (1868). This synthesis
was considered a great triumph of chemical investigation, and awakened
niany bright dreams for the future. Even then it was suggested that the
time was near at hand when foods could be produced practically by
synthetic methods. Although this hope has not yet been fully
realized, nevertheless, such syntheses as that of alizarin have an indirect
effect upon the production of foods because whenever a natural substance
is replaced by an artificial one a considerable amount of acreage is
released.

As far as the animal organism is concerned, the hexoses are the most
important representatives of the monosaccharides, and for a long time they,
and the corresponding polysaccharides, were the only carbohydrates to be
considered at all. It was not until 1892 that a sugar with five molecules of
carbon corresponding to the formula C5H10O5 was discovered. In that
year Jastrowitz ^ found a specimen of human urine showing strong reducing
properties but which fermented little if any, and was moreover optically
inactive. Salkowski' then showed that a pentose was present. In the
same year Kossel,* by the hydrolysis of yeast-nucleic-acid wth hydrochloric
acid, obtained furfurol, and soon afterwards Hammarsten ^ made similar
observations in studying the nucleoproteid obtained from the pancreas;
later on Salkowski " followed the matter still further, and proved finally
that pentoses are present in the above-mentioned products. Sugars,
then, of the five carbon series, have been detected one after another in
various products found in the body,' especially in the nucleoproteids. The

' See text-books on phannacology for the physiological and pharmacological action
of these substances.

' For other special cases, see van Rijn: Die Glykoside, Berlin, 1900.

' Zent. mod. \Vis.sensch. 19 and 35, pp. 3.37 and 593 (1892).

' " L'eber die Nucleinsjiure," Verhandl. physiol. Gesellsch, Berlin, and in Arch.
Physiol. .\nat. 1893, 1.57.

' Z. physiol. Cheni. 19, 19 (1894).

• Ibid. 27, 507 (1899).

' Z. klin. Med. 34, 160 (1898).

22 LECTURE II.

pentoses accordingly take part in the construction of animal tissue. The
only pentose which has been isolated from the organs and closely studied
is that from the pancreas-proteids, which C. Neuberg ' has identified
as /-xylose with the following configuration:

0 OH H OH
HC— C— C— C— CH2OH
H OHH

It belongs, therefore, to the aldoses.

Recently Wohlgemuth ^ has isolated the same pentose from liver-pro-
teid.' According to other investigations, it appears that the pentoses in the
animal organism are limited to the class of nucleoproteids, and in fact
that the carrier of the pentoses is not the albumin, but rather the other
component of the compound protein. This was shown, namely, by the
experiments of Ivar Bang,'' who succeeded by warming pancreas-nucleo-
proteid with dilute caustic potash in decomposing it into albumin and into
a product free from albumin, the so-called guanylic acid. This latter con-
tains the Z-xylose. Guanylic acid is decomposed by boiling with mineral
acids into guanine, glycerol, and pentose; it may therefore be considered
as glycerophosphoric acid in which the hydroxyl groups are partly re-
placed by guanine and partly by pentose. As to whether the pentose is
similarly combined in the other nucleoproteids has not yet been established.

The quantity of pentoses contained in the separate organs varies greatly
and depends directly upon the amount of nucleic substances present.
Grund ^ estimates the percentage of pentoses (calculated as xylose) present
in certain organs as follows:

Pancreas 2 48

Liver 0 .56

Thymus 0 56

Submaxillary Gland 0 53

Thyroid Gland 0 50

Kidneys 0.49

Spleen 0 46

Brain 0 22

Muscle 0.11

The values represent the
percentage of pentose (cal-
culated as xylose) in the
dry substance.

As has already been mentioned, the first pentose was discovered in
urine and formed by metabolism in the human organism. The disease

' Ber. 35, 1467 (1902).

= Z. physiol. Cham, 37, 475 (1903).

' For further particulars and other literature, see Neuberg in Ergeb. Physiol.
(Asher and Spire), 3, Abt. I, p. 37.3.

« Z. physiol. Chem. 31, 411 (1900-1901).

' Ibid. 35, 111 (1902). See also Bendix and Ebstein: Z. allg. Physiol. 2, Heft
1 (1902).

CARBOHYDRATES. 23

causing the elimination of this substance is called, if we adopt the suggestion
of Sallvowski, pentosuria. Up to the present time about a dozen cases
are known. Pentosuria is distinctly different from true diabetes. It
exists for years without showing the clinical indications of the latter
metabolic disturbance. In rare cases the elimination of pentose has been
detected in diabetes also,' but it is not known that there is any connection
between the two diseases. The amount of pentose eliminated varies in
individual cases between 0.2 and 1 per cent.

It is a striking fact that the pentoses eliminated in urine are, as a rule,
optically inactive. Luzzato,- alone, has described an optically active
pentose. The question next arises as to the source of pentose in urine.
First of all, Bial and Blumenthal ' have proved that pentosuria is inde-
pendent of the composition of the diet and especially as regards the amount
of pentoses in it. They have also shown that with persons afflicted with
the disease the combustion of carbohydrates, and thus also of /-arabinose,
is perfect. Pentoses in urine, therefore, do not find their source in the
food. The next possibility which would suggest itself is that perhaps
they arise from the breaking down of tissue and especially of the
nuclei. If this supposition were correct, we should expect that the pentose
found in urine would be the same as that found in the organs, namely,
inactive xylose. This now is not the case, as Neuberg * has shown, for
the pentose in urine is arabinose, and curiously enough almost always the
inactive, racemic form. Furthermore, this pentose for the most part does
not exist free in the urine, but is combined with urea. For the present, we
can only formulate hypotheses concerning its formation. Above all, we
are ignorant as to why the pentose should nearly always be eliminated in
an inactive form.

The five-carbon sugars are much more widely distributed in the vege-
table kingdom than in the animal. Up to the present time they have
not been identified with certainty in a free state, but on the other hand
they appear to be deposited in the nucleoproteids of certain plants in tlie
same manner as in animals, for Osborne and Harris ^ have isolated tritico-
nucleic acid from wheat flour. This manner of occurrence is inappreciable
as compared with that of high molecular pentosans, i.e., polysaccharides
of the pentoses. These substances are extremely widely distributed, and
take part in various ways in the building up of plants. By their hydrolytic
cleavage the simpler members of this series are obtained. Not only the
pentoses are found as pentosans, but we have methyl-pentosans as well

' Kiilz and Vogel: Z. Biol. 1895, 185.
' Hofmeister's Beitrige, 6, 87 (1904).
' Deut. med. Wochen.schr. 22 (1901).

* Ber. 33, 2243 (1900).

* Z. physiol. Chera. 36, 85 (1902).

24

LECTURE II.

(especially the polysaccharides of fueose), which are found everywhere in
sea-moss. The fueose pentosans are furthermore found in gum-arabic,
cerasin, and gum-tragacanth, in the leaves of plane and linden trees, in pine
and beechwood, etc. Therhamnoses, first found in quercitrin, the coloring
principle contained in the bark of dyer's oak {Quercus tintoria), are also
widely distributed. Most pentosans, however, are derived from the
simple pentoses. The most important of these are /-arabinose, which is
obtained from different gums; andZ-xylose, also called wood sugar because
it is the most important mother substance of lignin (xylogen). Xylogen
is also found in oat-, rye-^ and wheat-straw, etc. The pentosans in
general are by no means simple compounds, and yield on being sub-
jected to hydrolysis all sorts of different sugars of the five and si.x carbon
series. Doubtless there are a great many intermediary products lying
between the simpler and more involved complexes. As regards their
physiological function in the plant organism, our knowledge is still very
limited. We shall see later on that they are of importance for the
nourishment of animal organisms, particularly the herl)ivora. The fol-
lowing table will give some idea concerning the occurrence of pentosans,
the values being given in terms of pentose:

Meadow hay . .
Molasses fodder
Rape cake . .
Turnip ....
Oil-.seed cake .
Acrospires . .
Rice flovir . . .

Per cent.

21

64

15

98

11

50

2

26

9

07

14

12

5

73

Bruised barley
Sesame cake
Table turnip
Spinach . . .
Sauerkraut . .
Coffee beans

7.96
3.87
1.13
1.02
0.96
6.5

As regards the formation of pentoses in plant organisms, we have no
experimental data. Chemically, we can, as has already been mentioned,
easily account for it in three ways. The simplest explanation is that of
the formation from formaldehyde, which is hypothetically the first assim-
ilation product of the carbonic acid in the air by the green leaves. Five
molecules of the aldehyde will condense to form one molecule of pentose
(5 X CHoO = C5H10O5). It is also conceivable, that glycerose obtained
by the oxidation of glycerol is the starting-point, and from thence by the
third method of building up a sugar, namely, the addition of carbon
atoms, the pentoses may be formed. Finally it is possible that the
pentoses are formed by the breaking down of higher sugars. At all events
this relatively simple class of chemical compounds shows to what ex-
tremely diverse purposes the vegetable organism is capable of building
itself up.

CARBOHYDRATES. 25

We know absolutely notiiing with regard to the formation of sugars of
the five carbon series in the animal organism and concerning their signifi-
cance in the organism. It would seem most likely that the source of their
presence is to be sought in the diet, although such a relationship has not
yet been definitel}' traced.

We now come to that group of monosaccharides which is most important
for the animal organism, namely, the hexoses of the general formula
C6H12O6. We have already seen that, according to the rule of Le Bel and
van 't Hoff, there are sixteen possible isomeric aldehydes having this gen-
eral formula. We need consider here only the mannoscs, the glucoses, the
galactoses, and the fructoses. Before taking up these sugars in detail we
will mention some of the more important general reactions of sugars to
the extent necessary for us to understand the physiological behavior of
different kinds of sugars.

The simple sugars, in accordance with their aldehyde or ketone nature,
are very readily oxidized. They reduce, therefore, metallic oxides on
warming their alkaline solutions. Some of the qualitative and quanti-
tative methods of analysis, such as those of Fehling, Trommer, and
Bottcher, are based upon this property.

On heating a solution of sugar in caustic soda, or potash, a decomposition
takes place (Moore's test). The liquid turns brown, and among other
substances, lactic acid, catechol, and formic acid are formed.

If half a cubic centimeter of a dilute, aqueous solution of rf-glucose is
treated with a few drops of a ten per cent solution of «-naphthol in alco-
hol and then one cubic centimeter of concentrated sulphuric acid cau-
tiously added, the zone of contact becomes reddish violet in color. On
shaking, the mixture assumes this color (Molisch). This test is often
used for detecting the presence of sugar in proteins, etc., and depends upon
the formation of furfurol l_)y the action of the concentrated sulphuric acid
upon the sugar.

If a sugar solution is evaporated to dryness and the residue heated
somewhat, or if the sugar itself is at once exposed to direct heat, carboni-
zation takes place with a characteristic odor. The ma,ss is called caramel.

An important reaction, and one which has become of great consequence
in the investigation of the different kinds of sugars, is the combining of
many sugars with hydrazines in acetic acid solution, water being eliminated
and hydrazones formed. The most important of these compounds are
those with phenyl-hj'drazine.' If an approximately ten per cent aqueous
solution of glucose, for example, is treated with a solution of phenyl-
hydrazine in acetic acid and then heated on the water-bath for ten or
fifteen minutes, fine yellow needles are deposited whose composition

' Emil Fischer: " VerbindunRen dcs Phenylliydrazins mit don Zuckerarten," Ber.
17, 579 (1884), and 20, 821 (1S87).

26 LECTURE II.

corresponds to the formula C18H22N4O4. This compound is known as
glucosazo7ie.' It is formed by the action of two molecules of phenyl-
hydrazine upon one molecule of sugar, and in fact the reaction takes place
in two stages. First the sugar combines with one molecule of the base,
forming a hydrozone as follows:

CH2(0H) [CH(0H)]3CH0H . CHO + CgHsNH . NH2 =
CHaCOH) [CH(0H)]3 . CHOH : N . NH . CeHs + H2O.

* ^■
As this compound is very readily soluble in water, this phase of the
reaction is not noticeable in the reaction with glucose." Then on warm-
ing with an excess of phenyl-hydrazine, the alcoholic group marked with
an * undergoes a peculiar oxidation. The group is changed into carbonyl,
CO, and is thus capable of combining with a second molecule of phenyl-
hydrazine, to form an ozazone of the formula:

CH20H[CH(OH)]3 . C - CH
II II
CeHs . HN . N N . NH . CgHs

As we shall soon see, fruit-sugar (fructose) instead of being an aldehyde,

is a ketone with the following structure:

CHoOH

I
CO

I

HO— C— H

I
H— C— OH

I
H— C— OH

I

CH2OH
Now it is possible to obtain exactly the same glucosazone from
fructose as from d-glucose, but in this case the two stages of the reaction
take place in the reverse order; the ketone being first acted upon then by
oxidation an aldehyde is formed, which reacts with a second molecule of
phenyl-hydrazine, as shown by the following scheme:

CHzCOH) [CH(0H)]3 .C CHafOH)

II *

CeHs . HN . N

CH2OH . [CH(0H)]3 . C - CH

II II
CeHs . HN - N N . NH . CeHs

' Similarly we speak of galactosazone, arabinosazone, xylosazone, maltosazone, etc.
' With mannose a difficultly soluble phenylhydrazone is formed and can be isolated.

CARBOHYDRATES. 27

Natural and artificial sugars which reduce Fehling's solution (includ-
ing milk sugar and maltose) give the above reaction. The products
obtained are very characteristic, and are the most valuable means tliat
we possess for recognizing and separating the sugars.

An important property of the natural sugars which has been mentioned
already is their optical activity. Quantitative methods for the analysis
of sugars are based upon this property, and it serves for classifying the
sugars as well. Thus the dextro-rotary dextrose wjis first designated as
d-glucose, and la?vo-rotary laevulose as Z-fructose. Emil Fischer then
proposed a different method of nomenclature which shows the relation of
the compounds to one another. The monoses derived from a d-, I-, or
t-monose are also designated by the letters d-, 1-, and ?-, even when they
possess a rotary power opposite in sign to that indicated by these letters.
In this way laevulose is now designated as rf-fructose, because of its close
relation to rf-glucose, or dextrose.

The most interesting reaction of sugar from a liiological standpoint is
its ability to undergo fermentation. Thus different yeasts cause dextrose
to break down into alcohol and carbon dioxide (alcoholic fermentation) :

CsHisOe = 2 C2H5OH + 2 COo.

On the other hand, bacterium laciis converts it into ordinary lactic
acid (lactic acid fermentation) :

CsH.oOe = 2C3H6O3.

Finally dextrose may be converted into butyric acid, carbon dioxide, and
hydrogen by the action of certain microbes (butyric acid fermentation) :

CfiHiaOe = C4H8O2 + 2 COo + 2 H2.

At this place we shall not take up these processes in further detail. We
shall later on find opportunity for showing how great an influence the
stereo-configuration of the different sugars has upon their fermentability,
and how Emil Fischer by the aid of fermentation studies was able to
formulate hypotheses and arrive at conclusions which are of great
biological importance and form the foundation of our whole knowledge
concerning fermentation reactions.

It remains still to show certain relations between the sugars and two
other classes of compounds, namely, their reduction products (the corre-
sponding alcohols) and their oxidation products (the acids). The former
relation is at once apparent from the following summarj^: glucose is the
aldehyde of sorbite, mannose of mannite, and galactose of dulcite, while
fructose is the ketone of mannite. By oxidation we obtain:

From fjtftcose. first the monobasic gluconic acid, then by furtlier
oxidation the dibasic saccharic acid.

28

LECTURE II.

From mannose, the monobasic mannonic acid and then the dibasic
manno-saccharic acid.

From galactose the monobasic galactonic acid and then the dibasic
mucic acid.

Fructose behaves quite differently on oxidation. In the case of the
above-mentioned sugars, which are all aldoses, acids are obtained by
oxidation having the same number of carbon atoms as the original sugars.
Fructose, on the other hand, is a ketone, and on being oxidized breaks down
into compounds containing a smaller number of carbon atoms.

These reactions are naturally not peculiar to hexoses, and for the simpler
or higher monosaccharides there are corresponding alcohols as well as
monobasic and dibasic acids. Thus we have for example:

Bioses :

Alcohol
CH2OH

bugar
CHO

Monoliasie Acid
CO . OH

Dibasic Acid
CO . OH

Trioses :

CH2OH
Glycol

CHoOH

I
CHOH

CHoOH CH2OH

Glycolose Glycollic Acid

(Glycolaldehyde)

CHO

I
CHOH

CO . OH

I
CHOH

CO . OH
Oxalic .\cid

CO. OH

I
CHOH

CHoOH

Glycerol

Tetroses :

CHoOH

I
CHOH

I
CHOH

CH2OH CH2OH

Glyoerose Glyceric Acids

(Glyceraldehyde)

CHO

I
CHOH

CHOH

CO. OH

I
CHOH

CHOH

COOH
Tartronic Acid

COOH

I
CHOH

CHOH

CH2OH
d- and l-
Erythritol

CH2OH

d- and Z-
Erythrose

CH2OH

Erythric
Acids

COOH

Tartaric
Acids (4)

Erythritol was discovered by Lamy (1S52) in the algae Protococcus
vulgaris. It is optically inactive.

In the group of the pentoses, it has already been stated that arabinose
corresponds to the alcohol arabitol and the two acids, arabonic and 1-trioxy-
glutaric, while with xylose we have the alcohol xylitol and two correspond-
ing acids. Although these last alcohols and acids have up to the present

CARBOHYDRATES. 29

time not been found in nature, yet a native alcohol adonitol, obtained in an
optically inactive form from Adonis vernalis, corresponds to rhibose, a
sugar of the pentose series and which has onl}' been prepared synthetically.

In the case of the sugars containing more than six atoms of carbon and
which up to the present time have only been obtained artificially, the
seven-carbon alcohols perseitol and volemitol are found in nature. The
former is present in the unripe seeds, leaves, and pericarp of Persea gratis-
sima, while the latter is contained in Lactarius volemus, and has recently
been prepared from the rhizomes of several species of Primula.

Of the four above-mentioned hexoses, glucose, galactose, fructose and
aiannose, only the first three are found in the animal organism. d-Mannose
is found only in the vegetalile kingdom partly as such (for example, in
the sap of Japanese A morpho phallus Konjako), to some extent as glucoside-
like compounds (thus, strophantobiose decomposes into d-mannose and
rhamnose), and finally very extensively in anhydride-like, condensation
products known as mannans.

Fructose occurs similarly in the vegetable kingdom, and likewise either
free or combined. In the former state it is seldom found pure, but usually
is mixed with other sugars as a component of many fruits. Fructose is
formed, furthermore, by the hydrolysis of many vegetable substances ;
thus, of inulin, the reserve-substance "in the tubers of dahlia, helianthus,
sweet potato, elecampane, etc.

Its most important occurrence is in cane sugar, by the hydroh'sis of
which one molecule of (/-glucose and one molecule of d-fructose are
obtained. This mixture is known as invert sugar.

In the products of the animal kingdom, fructose is not often found. In
honey it occurs together with glucose. It is sometirnes to be found in the
urine after one has eaten considerable fruit. In rare cases it is found in
larger amounts in the urine of a diabetic. That fructose occurs normally
in animal tissues is e.xtremely doubtful.'

Grape sugar, d-glucose, glucose or dextrose, as it is variously called,
plays without question the most important role of all the monosaccharides
in the animal system. It is this form which carbohydrates in general
assume before absorption and assimilation. As rf-glucose the greater part
of the carbohydrate is conducted from organ to organ, from the place of
storage to the place of consumption. Glucose is always present in the
blood, and the amount varies only within narrow limits, averaging from
0.05 to 0.1 per cent in different animals.- These figures, however, are not
accurate, because glucose is not the onlv sugar that is found in blood. On

' Cf. .\dler and .Adler: Pfliiger'-s Arch. 110, 99 (190.5); Neuberg and Strauss: Z.
physiol. Chem. 36, 23.3 (1902); Paidolf Ofner: Monatsh. 25, 1153 (1904); 26, 11G5
(1905); and Z. physiol. Chem. 45, 359 (1905).

» See Lecture XXIII.

30 LECTURE II.

the other hand, it is not true, as Asher and Rosenfeld' have recently-
shown, that the greater part of the glucose, or perhaps all of it, is present
in a combined state. Glucose from blood diffuses through a parchment
membrane, even when the outer liquid is a blood deficient in sugar, made
so by the action of yeast. Glucose is also present in certain organs (mus-
cles). It is often hard to decide whether the glucose found was pre-formed,
or whether it has been produced secondarily from a carbohydrate of higher
molecular weight by hydrolytic fermentation. Normal human urine fre-
quently contains glucose, but always in very small quantities. It may
appear in larger amounts after a diet rich in carbohydrates, especially after
large amounts of grape sugar have been taken into the system. This is
spoken of as alimentary glucosuriar

The elimination of considerable quantities of sugar in the urine has
been observed after the introduction of numerous chemical substances into
the system, as, for example, strychnin, curari, phosphorus, etc.

The most interesting form of glycosuria is that produced by phloridzin
and known as phloridzin-diabetes. Phloridzin ' is a glucoside obtained
from the root-bark of apple, pear, cherry, and plum trees, and yields by
hydrolysis glucose and phloretin:

C2iH240,o + HoO = CeHizOe + CisHuOs

phloretin

The phloretin is decomposed further into phloroglucinol and phloretic
acid:

CisHuOs + H2O = C6H3(OH)3 + CsHioOa

phloroglucinol phloretic acid

We shall consider these artificially-produced glucosurias at another
place.

Glucosuria has been observed, furthermore, by Hofmeister * when he
fed starch to starved dogs. Bohm and Hoffmann ^ have described the
appearance of large amounts of sugar in the urine of cats which were
confined and protected from cooling off by coverings. Glucosuria can
also be produced by a cold.

Glucose is very widely distributed in the vegetalile kingdom partly as
such, partly in large storage deposits in the form of starch, and partly as

' Asher and Rosenf eld: Zentr Physiol. 19, 449 (1905).

2 Concerning sugar in urine, see Pfliiger, Schondorf, andWenzel; Pfliiger's Arch. 105,
121 (1904). It is to be remembered that chloroform, for example, when boiled in
alkaline solution shows a strong reducing power.

' J. S. Stass: Ann. 30, 192 (1840).

* Arch. exp. Path. Pharm. 26, 355 (1890).

' Ibid. 8, 271 and 375 (1878).

CARBOHYDRATES.

31

the framework-substance in all varieties of cellulose. Finally it takes
part in the formation of a great number of glucosides.

The last member of this series which we have to consider is galactose.
Up to the present time it has never been detected with certainty in the
free state. It is found as a constituent of certain vegetable glucosides,
for example, digitonin and sapotoxin.

On the other hand, numerous polymers of galactose, the so-called galac-
tanes, are known, some of which yield galactose alone on hydrolysis, while
some give other sugars as well.

In the animal organism, galactose is present chiefly in milk-sugar, but
similarly it has been obtained by the hydrolysis of cerebron (see page 20) .
We know very little concerning the formation of galactose or of milk-
sugar. There are, as has been mentioned, certain well-known higher
sugars in the vegetable kingdom which yield galactose, but it is very
questionable that there is any direct connection between the galactose in
the nourishment and that of milk-sugar. We shall come back to this
point in the discussion of the latter compound.

At this place we will consider two compounds which are very closely
related to glucose, namely, glucuronic acid and glucosamine (chitosamine).

Glucuronic acid (also written glycuronic) is a derivative of glucose.
Schmiedeberg and ]\Ieyer ' suspected this, for they realized that the com-
pound combined the properties of an acid, an aldehyde, and a p^olyvalent
alcohol. It was not proved, however, until Thierfelder ' succeeded in
changing glucuronic acid into <i-saccharic acid, and Fischer and Piloty^
effected its synthesis from rf-saccharic acid. By the work of the last
named, the configuration was established, as shown by the following
summary:

CHO

1

CO. OH

1

CHO

1

HCOH

1

HCOH

1

HCOH

1

HOCH

1

HOCH

1

HOCH

1

HCOH

j

HCOH

HCOH
HCOH

HCOH

1

HCOH

1

CH2OH

CO. OH

CO. OH

d-Glucose

c?-Saccharic acid

d-Glucuronic acid

- Z. physiol. Chem. 3, 422 (1879).
' Ibid. 11, 389 (1887).
' Ber. 24, 521 (1891).

32 LECTURE II.

Glucuronic acid itself is not crystalline, but on boiling its solution,
glucurone lactone, CeHgOe, crystallizes out:

CHO . CHOH . CH . CHOH . CHOH . CO.

\— 0

Glucuronic acid does not occur free in the animal organism, and the
uncombined acid has not yet been positively identified in the vegetable
kingdom. Its derivatives are always present to a slight extent in urine,
in the blood, and in the liver.' It has always been found present in ester-
like coupling with various compounds from which the glucuronic acid may
be prepared by saponifying reagents. In normal urine we find phenol-,
indoxyl-, and skatoxyl-glucuronic acids. These compounds are of much
less importance than those well-known compounds of glucuronic acid with
different substances introduced into the body. Glucuronic acid pairs with
members of the aliphatic series (alcohols, aldehydes, ketones, etc.) and also
of the aromatic series. The best known of these compounds are those with
camphor and chloral, although the number of conjugated d-glucuronic
acids that have been studied is very large.^ According to their entire
behavior they may be regarded as glucosides.

Up to the present time, the way in which glucuronic acid is formed has
not been clearly decided. Again, we know nothing regarding the amount
formed under normal conditions, for it is highly probable that the acid is
oxidized further in the organism when no compound capable of combining
with it is present. It would seem most probable that it is formed from
glucose, but, as Emil Fischer^ has stated, it is difficult to understartd a
direct transformation here. It is hard to see why in such a case the
oxidation should take place at the primary alcohol group rather than at
the readily-oxidizable aldehyde group. Emil Fisclfer assumes, therefore,
that by the introduction of substances such as camphor, there is an inter-
mediate combination with the glucose so that the aldehyde group is thus
protected from oxidation. Then by oxidation of the free primary alcohol
group, the glucuronic acid compound is formed.

Thierfelder * called attention to another source of glucuronic acid by
showing that when camphor and chloral hydrate are fed to hungry dogs
the corresponding conjugated acid is eliminated in the urine. It, therefore,

' Paul Mayer and Cad Neuberg: Z. physiol. C'hpm. 29. 2.56 (1000). Paul Mayer: ibid.
32, 518 (1901). Lepine and Boulud: Compt. rend. 133, 138 (1901); 134, 398 (1902);
141, 453 (1905).

'' Hildebrandt has recently studied a large number of the.se compounds. See sum-
mary in article by Neuberg on Pentoses and Glucuronic Acid, Ergeb. Physiol. (Asher
and Spiro), 3 Abt I, p. 373.

» E. Fischer and O. Piloty: Ber. 24, 521 (1891).

« Z. physiol. Chem. 10, ioS (1886).

CARBOHYDRATES. 33

seemed probable that the glucuronic acid was formed from decomposed
albumin. According to recent investigations concerning the reserve
stores of sugar in the starved organism, especially of glycogen, all such
conclusions have become doubtful. Furthermore, the whole question
of the formation of glucuronic acid from albumin coincides with that of
carbohydrates from proteins. At another place we shall discuss this
problem in detail. On the other hand, an oljservation made by Salkowski
and Neuberg' is worthy of mention here. They found that glucuronic
acid when exposed to intense putrefaction goes over into the aldopentose,
l-xijlose, with the splitting off of carbonic acid.

CHO COH

I I

HCOH HCOH

I I

HOCH HOCH

HCOH HCOH

I I

HCOH H.COH

I
CO. OH

d-GlucurcJnic acid /-Xylose

We have met with ^xylose before. It is found in the nucleoproteids of
the pancreas and liver, and is perhaps the sole pentose occurring in the
animal organism. This observed transformation connects the aldo-
hexose, glucose, with the aldopentose, xylose. It is indeed conceivable that
the formation of xylose in the animal system may take place in a similar
wa}'. For the present we have no precise knowledge about this process,
and we know just as little regarding the place of formation of glucuronic
acid, i.e., in what part of the body the compound is synthesized. It is
extremely probable that it is not limited to a single organ.-

Glucuronic acid is to be considered as a substance which protects the
organism against the action of various kinds of substances some of which
are formed in the body while some are brought into contact with the cells
of the body from the outside. It combines with these substances and
makes them harmless. We shall subsequently meet with other compounds
(glycocoll, sulphuric acid) which perform the same task. The poisons
thus neutralized are almost always substances which cannot be destroyed
in the organism by direct oxidation. Glucuronic acid may combine
directly with these poisons, that is, without the latter undergoing any

■ Z. physiol Chem. 36, 261 (1902).

' Julius Tohl Arch. exp. Path. Pharm. 41, 97 (1898).

34 LECTURE II,

other change. This is, for example, the case with substances containing
an hydroxyl group. Thus, trimethyl carbinol introduced into the system
is eliminated in the urine as a conjugated glucuronic acid:"

(CH3)3 . COH + C'eHioOy = (CH3)3 • CO.CeHgOe + H2O

Many compounds, however, are not chemically suitable for coupling
with glucuronic acid. They are prepared for combination by the action
of the animal body either by reduction, by oxidation, by hydration, or by
the two last-named processes acting together. Thus, for example, chloral
hydrate ^ and butylchloral hydrate ' are reduced. The former is changed
into trichlorethyl alcohol, and this combines with glucuronic acid, the
conjugated acid being eliminated in the urine. The compound formed is
known as urochloralic acid:

CI3C . CH + 2 H = CI3C . CH2OH + H2O,

CI3C . CH2OH + CeHioOy = CI3C . CH2O . CgHgOe + H2O.

A preliminary oxidation takes place in the case of o-nitrotoluol * it being
changed in the organism of the dog to nitrobenzyl alcohol:

NO2 . C6H4 . CH3 + O = NO2 . C6H4 . CH2OH,
NO2 . C6H4CH2OH + C'eHioOv = NO2 . C6H4 . CH2O . CfiHgOs + H2O.

Many camphor varieties undergo a similar preliminary oxidation.

In other cases the animal organism causes the poisonous substance to
take on water, and oxidation may take place simultaneously. An example
of this is found in the transformation of thujone into thujone hydrate,^
which then unites with glucuronic acid:

OC10H17OH + CeHioOy = OCioHijO . CeHgOe + H2O.

Camphene ' is changed into camphene glycol:

C10H16 + O + H2O = HO . CoHie . OH.

As the above equations show, the fundamental principle of the coupling
is in all cases the same, except that sometimes the poison can unite directly
with the glucuronic acid (or glucose — see above) , while otherwise this

' Thierfelder and von Mering: Z. physiol. Chem. 9, 511 (188.5).

' von Mering: ibid. 6, 480 (1882). von Mering and Musculus: Ber. 8, 662 (1875).

' Kulz: Pfliiger's .A.rch. 28, 506 (1882); 33, 221 (1883).

* Jaff^: Z. physiol. Chem. 2, 47 (1878-79), and Ber. 12, 1092 (1878).

' Emil Fromm and Hermann Hildebrandt: Z physiol. Chem. 33, 579 (1901).

« Fromm, Hildebrandt, and Clemens: ibid. 37, 189 (1902-03).

CARBOHYDRATES. 35

union is possible only after the animal organism itself has produced some
change in the objectionable substance.'

Quite different from glucuronic acid in its properties is the compound
d-glucosamine, idso known as chitosamine. It was firel prepared in large
amounts from the chit in of lobster shells," and later, as has been mentioned,
from mucin substances and as a cleavage product of proteins. The con-
stitution of glucosamine has been recently cleared up by Fischer and
Leuchs.' It is to be regarded as a derivative of either d-glucose or d-
mannose, in which the hydroxyl of the a position is replaced by the amido
group, NH2. Its configuration is the following:

H H OH

I I I

CHzCOH)— C — C — C — CH(NH2).CH0

I I I

OH QH H

Glucosamine is a very interesting compound. It forms an interniediate
step between the hexoses and the hydroxy-a-amino acids, which we will
soon meet with as cleavage products of the proteins, so that glucosamine,
in a sense, forms a bridge between the proteins and the carbohydrates.
At present we know nothing concerning the physiological significance of
glucosamine. It does not occur free in the above-mentioned substances,
but in a polymeric form either alone or with other sugars.

Finally there remains one other amino-sugar to mention which we have
already touched upon, namely, galactosamine. It was discovered by Schulz
and Ditthorn and represents a component of the glucoproteids in the albu-
minous gland of the frog. Its constitution is not known at present.

' Cf . Fromm's Die chemischen Schutzmittel des Tierkorpers bei Vergiftungen, Strass-
burg, 1903.

' Ledderhose: Z. physiol. Chem. 2, 213 (1878-79); 4, 139 (1880). H. Steudel: ibid.
34, 353 (1902).

' Ber. 36, 3787 (1902); 36, 24 (1903).

LECTURE III.

CARBOHYDRATES.

II.

Polysaccharides.

The polysaccharides, or compound sugars, which we now have to
consider, can Ije regarded, as we have seen, as glycosides of sugar itself; in
other words, they are formed from simpler sugars with elimination of
water; and, conversely, by the action of hydrolyzing agents (chemicals or
ferments), they may be broken down into their separate components, i.e.,
simple sugars. In discussing the monosaccharides, we often found oppor-
tunity to point out how widely distributed these complicated sugars are,
for the simple sugars themselves in some cases only occur in nature in this
state. Biologically this group assumes a distinctive position. The animal
and vegetable organisms store their reserves of carbohydrates in this
form. On the other hand, many representatives of the class may be
looked upon as the intermediary products between the more complicated
and simpler sugars, and owe their origin to a progressive, spontaneous
hydrolysis. In the center of all these processes of transformation taking
place in the animal organism, we find the hexoses, especially glucose;
whether a complicated sugar molecule such as starch breaks down, or
whether such a one as glycogen is formed, for example. In the plant
organism the relations are to some extent similar, except that here, as has
been previously mentioned, the sugars of the five-carbon series are more
common. It is, however, still an open question as to whether the
simple pentoses here take such a central position in the metabolism
of carbohydrates as the hexoses in the animal system, for up to the
present time the pentoses are known almost exclusively in the form of
polysaccharides (pentosans, etc.), concerning the formation of which our
knowledge is still very limited.

The group of polysaccharides is subdivided, according to the number of
sugar molecules which enter into their composition, into di-, tri-, tetrasac-
charides, etc., and the true polysaccharides.

The disaccharides ' consist of two molecules of the simple sugar minus

' Here only the hexobioses are considered, i.e., those composed of two molecules of
hexose. There are also bioses built up of sugars containing fewer carbon atoms, for
example, gluco-apiose, which is built up from /?-oxymethyl-tetrose (apiose) and glucose,
and is obtained from the glucoside in Petroselinum apiin. Again, we have the mano-
rhamnoses prepared from strophanthin.

36

CARBOHYDRATES. 37

one molecule of water. This conception corresponds to the empirical
formulas :

.C12H22O11 = (C6Hi206)2 - H2O or C12H02O1, + H2O = 2 (CeHisOe).

They correspontl in tlieir behavior to the monosaccharides. Cane-
s'ugar is an exception to this rule, as its alkaline solution is not capable of
reducing metallic oxides, whereas the other disaccharides retain entirely
the properties of the aldehj-de alcohols. The exceptional behavior of
cane-sugar has been ascribed to a protected position of its aldehyde and
ketone groups.

To this gi'oup belong sugars which are found in nature as such, besides
other sugars which arc formed I13' cleavage from sugars of liigher molecular
weight. Cane-sugar, milk-sugar, and maltose are of especial importance,
whereas the remaining members of the group are, according to their
occurrence and importance, of only limited interest. Worthy of men-
tion are trehalose, first found in ergot of rye; gentiobiose, obtained from
gentianose by partial hydrolysis produced bj- invertin or very dilute
sulphuric acid; and cellos^ (cellobiose), which is believed to stand in the
same relation to cellulose as maltose to starch, but has not j'et been found
in the vegetable kingdom. Melibiose is another hexobiose, and is formed
by partial inversion of either melitriose or of raffinose, either by the action
of dilute acids or by certain varieties of yeast.

Before discussing the above-mentioned, more important disaccharides,
we must mention two other hexobioses which have been obtained in a
peculiar manner, namely, isomaltose and isolactose. The former was
synthesized bj- Emil Fischer from grape-sugar by the action of cold, fuming
hydrochloric acid, and is said to be formed, on the other hand, together
with maltose by the breaking down of starch. It must be mentioned,
however, that the identification of isomaltose in most cases has not been
entirely satisfactorj', so that we are still unable to tell much about the
building up and breaking down of carbohydrates in plant and animal
organisms, and especially because it seems probable that quite a number
of different products have Iieen designated as maltose by various inves-
tigators. Isomaltose excited the interest of biologists in particular when,
in 1808, \. C. Hill ' succeeded in olitaining it from grape-sugar by the aid
of the maltogluca.se of yeast, and likewise4n' the so-called taka-diastase
from Asjiergillus oTjizae. Hill added the ferment to concentrated solutions
of grape-sugar, and showed that the fermentation reaction was to some
extent a reversible process. Hill himself regarded the product formed

' J. Chem. Soc. 73, 634 (1898); Ber. 34, l.JSO (1901); Proc. Chem. Soc. 19, 99 (1901);
17, 184 (1901).

38 LECTURE III.

as maltose. Emmerling ' showed, however, that isomaltose was chiefly
present in this case. Later on we shall take up the syntheses produced
by the action of ferments more in detail, as well as their biological impor-
tance.^

Isolactose occupies a quite similar position, and has been obtained by
Fischer and Armstrong ' from a mixture of d-glucose and d-galactose under
the influence of lactoglucase.

Cane-sugar, also known as sucrose, saccharose, and saccharobiose, is of
great importance for plant and animal organisms.* It plays an important
part in the reserve-stores of all Phanerogamia, and is found chiefly in
tissues containing no chlorophyll, although it is present in smaller quan-
tities in all parts of the plant. It occurs to the greatest extent in the
stalk of the sugar-millet (sorghum) and sugar-cane, in the sap of certain
kinds of palm, that of the sugar-maple, the birch and the carob tree (St.
John's bread). Considerable amounts are found in the ripe fruits and
leaves of various growths. At present the sugar-beet is cultivated exten-
sively on account of the cane-sugar it contains, and, together with the
sugar-cane, forms the source of practically all commercial sugar.

This important food and condiment has never been positively identified
in the animal organism. It is certain that it takes no part in intermediary
metabolism. This follows from the fact that cane-sugar introduced into
the veins is not utilized, but passes off unchanged in the urine. In order
for this sugar to be of value to the animal organism, it must first be
subjected to hydrolysis in the digestive tract. ^

Cane-sugar, as proved by Liebig in the year 1834, corresponds in its
composition to the formula C12H22O11. It decomposes under the action
of hydrolytic agents into one molecule of d-fructose and one of f/-glucose.
Since the d-fructose in this mixture rotates the plane of polarized light more
to the left than rf-glucose does to the right, the product is Isevorotary, that
is to say, in the opposite direction as compared with cane-sugar, which is
strongly dextrorotary. For this reason this mixture of equal parts of the
two hexoses obtained by the cleavage of cane-sugar is called invert-sugar,
and the process is spoken of as inversion." Its formation was first studied
by Dubrunfaut ' in 1830. Mixtures of fruit- and grape-sugars, moreover,
occur very extensively in nature (honey, fruit, etc.).

■ Ber. 34, 600 and 2206 (1901).

^ See lecture on Ferments.

5 Ber. 35, 3144 (1902).

' E. Schulze and S. Frankfurt; Z. physiol. Chem. 20, 511 (1895).

' Claude Bernard: "LeQons sur le Diabete," p. 249 (1877). Fritz Voit: Deut. Arch,
klin. Med. 58, 523 (1897).

' This term is also used in general to denote the hydrolytic decomposition of com-
pound carbohydrates into simple sugars. Tlie opposite change is called reversion.

' Compt. rend. 25, 308 (1847); 29, 51 (1849); 42, 901 (1856).

CARBOHYEiRATES. 39

Milk-sugar, also called lactose or lactobiose, occurs similarly in nature,
and was described as long ago as 1615 by Fabricio Bartoletti in the
" Encyclopaedia dogmatica," and described in 1700 by Testi and in 1715
by Vallisneri as a newly-discovered medicine. Milk-sugar is found in vary-
ing amounts in the milk of all mammals. During confinement it is often
found in small quantities in the urine.' Similarly in calving it has been
detected in the urine for several days before and after the birth. Again
after weaning, sugar is wont to pass off through the kidneys. Recently,
Porcher - has carefully studied the origin of the lactose in milk. He found
that extirpation of the breast-glands of milch-goats and cows soon caused
a marked increase in the amount of sugar in the blood, while, at the same
time, glucose appeared in the urine. These experiments make it seem
very probable that the milk-sugar is first formed in the breast, and appar-
ently from glucose alone, and not out of the glucose and galactose in the
food

Milk-sugar has never been found in the vegetable kingdom.' On
being subjected to hydrolysis it breaks up into one molecule of glucose
and one of galactose. By oxidizing it with nitric acid, mucic acid,
COOH . (CH0H)4 . COOH, is formed.

Maltose, also called malt-sugar, maltobiose, ptyalose, and cerealose,
occupies a quite different position from the above two disaccharides. It
is a cleavage product of starch, and in fact an intermediary product which
usually is immediately hydrolyzed further as fast as it is formed. It is
true that small amounts of maltose are met with now and then in plant
organisms, and it is quite possible that it is here also a transitory product
in the metabolism of carbohydrates. Recent investigations make it seem
probable that maltose is also found as a glucoside in the vegetable kingdom.

In animal organs (liver, blood, etc.) maltose has been repeatedly found,
although always in small amounts; and furthermore, in many cases the
methods of identification have not been entirely satisfactory. The most
important manner of formation is by the action of a ferment upon starch.

.\s long ago as 1785 Irvine, and in 1815 Kirchhoff,* observed that extract
of malt was capable of breaking down starch. The sugar formed was
recognized first by Dubrunfaut ^ in 1822. The active principle in malt,
the so-called diasta.se, was first isolated by Payen and Persoz." Starch

■ a. Franz Hofraeister; Z. physiol. Chem. 1, 101 (1877-78), P. Kaltenbach: ihid.
2, 360 (1878-79). F. \. Lcmaire: ibid. 21, 442 (1895-96).

■ Ch. Porcher: Conipt. rend. 141, 73 and 467 (1905).

' Bouchardat [Compt. rend. 73, 462 (1871)] claimed to have found milk-sugar in the
ripe fruit of achras sapola, hut tliis has not been confirmed.
< Schweigger'.s J. 15, 389.
' .\nn. chim. phys. 3, 21 and 178.
• Ibid. 2, 53, 56, 73, and 337.

40 _ LECTURE III.

•
does not decompose into maltose alone, but quite a number of other prod-
ucts are formed at the same time. The whole process of dissolving the
starch by the action of diastase has been made the subject of countless
studies. A large number of intermediate products have been isolated
and provided with special names, but it would be out of place to discuss
here all the transformation products that have been described, for their
manner of formation and their chemical characteristics are not yet accu-
rately known. The reason for this is mainly that we are in doubt con-
cerning the homogeneity of the starting material, the starch itself, and
know still less concerning diastase.

Ferments, corresponding in their action to this malt-diastase, are widely
distributed in nature, and take an important part in the metabolism of
carbohydrates in plants. They give back to the metabolism of the plant
its reserve-stores, the insoluble starch.

The animal organism, as well, is known to contain ferments which dis-
solve starch and convert it into sugars, and in this process maltose is
formed as an intermediate product, which then breaks down into two
molecules of grape-sugar. Later on we shall have to consider such trans-
formations in detail. It may be mentioned here, however, that in the
breaking down of glycogen, the stored carbohydrate of the animal system,
maltose has also been observed.

Polysaccharides which are anhydrides of three and four sugar molecules
are also known and have been accurately described, while in the case of the
more complicated compound sugars we know nothing at present concern-
ing the number of sugar molecules which take part in their formation.
We know of a trisaccharide, rhamninose, which is composed of two pentoses
and one hexose; this is obtained in the decomposition of a glycoside
obtained in the fruit of Rhamnus infectoria, the xanthorhamnin. Rham-
ninose breaks down into two molecules of rhamnose and one molecule of
d-glucose. Trisaccharides composed of three molecules of hexose are
more widely distributed in nature. Of these we will mention raffinose
(also known as melitriose or gossypose) which is found in different plants
and in the sugar-beet. A tetrasaccharide, stachyose, (manna-tetrasac-
charide), is known, and was first obtained from the manna of ash. On
being treated with dilute mineral acids it takes on water, and is decom-
posed into one molecule of d-fructose, one of d-glucose, and two of
d-galactose.

The higher polysaccharides have been studied but little. We know
merely that the complete hydrolysis of these compounds gives mono-
saccharides as final products. We know nothing, however, concerning
the number of molecules of simple sugar which take part in their forma-
tion. To the widely different substances of this large group the. general
formula (CeHioOs)., is given, which signifies that the compound is com-

CARBOHYDRATES. 41

posed of x-molecules of sugar anhydrides. The attempt has frequently
been made to determine the molecuhir weight of many of these compounds.
Thus with starch, the formula derived in this way has been given as
CisHaoOio on the one hand, and as C300H600O300 on the other. We will
meet with the same difhculty when we come to study the proteins.

The following substances belong to this group: starch, inulin, cellulose,
gimis, vegetable mucilages, and glycogen. They are, with, the possible
exception of glycogen and inulin, all known only in the amorphous state.
Water dissolves some of them completely, others merely swell, while the
remainder are unaffected. The solutions do not taste sweet, but are
optically active. In general they will not diffuse through a parchment
membrane, and for this reason they are also called saccharo-colloids.
Chemically they are indifferent compounds, and will not combine, for
example, with phenj-l-hydrazine. With the exception of dextrin, they will
not reduce metallic oxides in alkaline solution.

These various higher poh'saccharides differ widely in biological signifi-
cance. Thus starch and glycogen, which on account of their similar
nature may be designated as vegetable and animal glycogens, are found
to be the most important reserve-substances of the carbohydrate group
that occur in the vegetable and animal kingdoms respectively. Inulin
has a similar nature. The gums and vegetable mucilages, on the other
hand, fulfill an entirely cUfferent purpose. They serve, at least to some
extent, to close up injuries, and correspond to the wound-secretions of
animals. Then again, those substances classed together under the name of
cellulose have a still different significance. They are found extensively
in the vegetable world, and form in general the chief constituents of the
walls of plant cells; or at least this is true from the mosses and ferns up
through the whole order of phanerogams, while in the studies concerning
bacteria, fungi, and alga;, the conclusions drawn have not been uniform.'
A peculiar position is occupied by the dextrins, which it is now certain
are not individual substances, but very complicated mixtures. As we
have already seen, they are to be looked upon as the decomposition
products of starch.

Now, after this brief introduction, we shall turn our attention to the indi-
vidual representatives of this class. Sharply distinct in its entire behavior
from all the other higher polysaccharides, is cellulose. It is perfectly insol-
uble in the ordinary solvent.s, water, alcohol, ether, etc. There is, in fact,
only one good solvent known for cellulose, and this is an ammoniacal
solution of copper oxide (Schweitzer's reagent). If cellulose is treated
with concentrated sulphuric acid at ordinary temperatures, first of all the
sulphuric acid ester of cellulose is formed. If this sulphuric acid solution

' For the chemical composition of the cell membranes of different cryptogams, see
Karl Muller: Z. physiol. Chem. 46, 2G5 (1905).

42 LECTURE III.

is diluted with water and boiled, glucose is formed. Cellulose is found
almost exclusively in tiie vegetable kingdom. In the animal world it has
only been identified with certainty in the shells of the tunicata.' In the
cell walls of plants there are found not only sugars of the cellulose group,
but other complicated carbohydrates as well, which, on being subjected to
hydrolysis, sometimes yield glucose, sometimes no glucose at all, besides
other sugars (arabinose, xylose, etc.). These substances have been
designated by Schulze as hemicelluloses.- In building up the cell walls,
furthermore, the pentosans, which yield only pentoses on hydrolysis, also
take an active part.

As is well known, the cell walls undergo changes with age which at first
are manifest externally only by greater rigidity. We speak of Ugnification.
This process has been made the subject of much careful investigation
without ever being clearly explained. Erdmann ' assumes that " wood "
is formed from cellulose by its combination with other substances which
are perhaps of an aromatic character.''

Exuding from the various tissue-complexes (medullary-, wood-, and
bark-parenchyma) of the cell membranes come the different gums. They
are very widely distributed in nature, and, by breaking them down with
dilute acids, usually galactose and arabinose are formed. Naturally this
group cannot be regarded as homogeneous. Especially well known are
gum-arabic and cherry-gum. To this class belongs agar-agar (obtained
from East- Asiatic alg:^), which has l^ecome important as a culture medium
for bacteria. Again, the common vegetable mucilages are included, being
different from the gums onh' by their insolubility, or difficult solubility,
in water.

We now come to those members of the carbohydrate group which the
animal and vegetable organisms temporarily withdraw from the general
metabolism in order to be alile to make use of them at any time by trans-
forming them back into simple sugars. We have, in cane-sugar, already
met with such a reserve-substance for plants. At least an equally impor-
tant part is taken by the starches ^ (amylum) which are found in the
seeds, roots, bulbs, tubers, pith of trees in winter (especialh^ in vegetation
robbed of their leaves at this season of the year) , etc. The amount of
starch present in some of these stores may amount to even eighty per cent
of the dry substance. Amylum occurs in the form of stratified granules.

' C. Schmidt: J. pr. Chem. 38, 43.3 (1846). Franchimont: Ber. 12, 1938 (1879).
Winterstein; Ber. 26, 362 (1893).

'' Schulze, Steiger, and Ma.xwell: Z. physiol. Chem. 14, 227 (1890). Schulze: ibid.
16, .387 (1892); 19, 38 (1894); Ber. 22, 1192 (1889), and 24, 2271 (1891).

' J. Erdmann: Ann. Suppl. 5, 223 (1867).

* Cf. Viktor Grafe: Monatsh. 25, 987 (1904).

* Cf. Brown and Heron, Ann 199, 1G5 (1879).

CARBOHYDRATES. 43

differing in form and size with different plants. The concentric rings
represent its gradual growth. Starch is scarcely changed at all by cold
water, but warm water makes the grains swell up and finally burst, form-
ing starch-paste. Starch-paste does not reduce metallic oxides in alkaline
solutions. A very rapid swelling is brought about at ordinary temper-
atures by means of concentrated solutions of metallic salts. Even with
dilute alkalies a starch-paste may be prepared in a short time. A well-
known test for starch is the indigo-blue coloration produced by iodine
solutions in the presence of hydriodic acid or an iodide. The color-
aUon is not permanent on boiling, but reappears on cooling. The
presence of substances capable of being o.xidized by iodine (caustic alkali,
sulphurous acid, arsenious acid, etc.) will prevent the appearance of this test,
the blue color only being obtained when such impurities have been oxidized.
All varieties of starch do not give a blue color with iodine; some of them
give a reddish-brown color, and with others the color is that of red wine.

At present we do not know a great deal concerning the significance of
these different colorations; it is positively certain, however, that starch
cannot be regarded as a chemical individual. The conception " starch "
comprises a large group of substances of similar physical and chemical
properties, which form a unit on account of their common biological signifi-
cance. An attempt has been made to get an idea concerning the structure
of starches by studying their decomposition products. On boiling them
with dilute acids, glucose is obtained. If the acid is allowed to act in
the cold, or with only gentle heating, a hydration product is produced
which is known as " soluble starch." By the action of cold, dilute mineral
acids for several weeks, or bj' an hour's treatment with 4 per cent, sul-
phuric acid at 80° C, the so-called " amylodextrin " is obtained, and,
on further hydrolysis of the latter, dextrins are formed, while, as just
mentioned, the final product is grape-sugar. We stated in connection
with malto.se that a similar breaking up of the starch molecule could be
effected liy ferments, in this case diastattc ferments. It was also men-
tioned then, that at present we are not able to deduce a picture of the
starch formation from a study of the great number of intermediate products
obtained by partial hydrolysis and designated in the literature with par-
ticular names. We mu.st be satisfied, for the time being, with the knowl-
edge that amj-lum contains a large number of anhydride-like grape-sugar
molecules, and bj- taking on water it is decomposed, step by step, into
smaller molecules, and finally into the basal component glucose. We
shall find, later on, that the proteins are quite similarly constituted.
Soluble starch, amylodextrin, dextrin, etc., correspond to the albumoses and
peptones, while glucose, the elementary building material, corresponds to
the amino-acids. A similar analogy is found with the fats, although here
the relations are much simpler.

44 LECTURE III.

In discussing fruit-sugar we met with a reserve-carboliydrate, inulin,
which in its biological relations completely corresponds to starch. It
is found in the roots of Inula Helenium, the bulbs of dahlias, etc., and is
different from starch in so far as it yields on hydrolysis fructose instead
of glucose. Furthermore, inulin dissolves in warm water without forming
a paste; iodine colors it yellow, and diastatic ferments do not attack it.

Finally, in many lichens, especially in Iceland-moss, another kind of
starch is recognized which likewise is colored yellow by iodine ; it dissolves
in hot water, and yields glucose upon complete hydrolysis. This is lichenin,
and, like inulin, it is not effected by diastatic ferments.

Besides these carbohydrates which are found in the reserve-stores of
plants, others, such as amylin, lavosin, cerosin, and secalin, are found in
grain-seeds. These substances yield, as a result of hydrolytic decom-
position, sometimes glucose and sometimes fructose. In the constructive-
tissue of Lupinus luteus is found galactin, a carbohydrate of this group
yielding only galactose upon complete hydrolysis. . Again, in the class of
graminea?, palms, liliaceie, amaryllidacece, irideae, and also in the many
dicotyledons, we meet with the so-called reserve-celluloses. We under-
stand by the term reserve-carbohydrates, substances which appear as solid
deposits on the cell-membrane of constructive tissue in seeds.
■ The dextrins are usually regarded as forming a particular class in the
group of carbohych-ates. They are, as has been stated, decomposition
products, and are obviously mixtures of substances with different molecular
weights. They form a transition stage between the " reserve-carbohy-
drates " and the " metabolic carbohydrates." By further hydrolysis
they yield molecules of glucose.

Glycogen is to the animal organism what starch is to plants. We have
to thank the French scientist Claude Bernard ' for its discovery, who in
1848 noticed the high sugar content of liver, and found sugar only absent
from it after prolonged starvation. A few years later, the same inves-
tigator succeeded in showing that the sugar observed in liver was not
directly present as such, but was formed gradually from a preliminary
state. He established the fact that by taking the liver from a dog right
after it is killed, the blood being washed off and the washing continued
in running water for forty minutes, then the last wash-water will no longer
show the presence of sugar. Even if a piece of such liver is boiled in
water, no sugar will be dissolved out of it. If, however, the liver is allowed

' See Bernard and Barreswil: Compt. rend. 27, 514 (1848); also E. F. W. Pfliiger:
"Das Glykogen und seine Beziehungen zur Zuckerkrankheit," Bonn, Martin Hager
(1905) and Max Cremer: " Physiologie des Glykogens," in Ergeb. Physio!. (Asher und
Spiro) 1, 80.3 (1902), Wiesbaden, published by T. F. Bergmann. A complete summary
of Bernard's work is found in "L'ceuvre de Claude Bernard," Paris, T. B. Bailliere
et Fils.

CARBOHYDRATES. ^ 45

to stand for twenty-four hours, a considerable quantity of sugar will then
be found present. This suggested to Bernard that a substance must be
present in liver which is dif&cultly soluble in water, but yielded sugar by
the action of the liver substance, and this must be " living," as the fol-
lowing experiment showed. After thoroughly washing the liver, half of
it was boiled, and from this it was not possible to obtain any more sugar,
whereas sugar was slowly formed in the other piece. Bernard not only
assumed the presence of a complicated substance in the liver from which
the sugar was formed, but he actually succeeded in isolating such a sub-
stance.' The method employed by him for the preparation of glycogen
is essentially the same as that of to-day. It depends upon the fact that
alcohol precipitates glycogen from an alkaline solution of the organ. By
dissolving it again in caustic potash and reprecipitating by alcohol, it is
easy to purify the crude glycogen. In this way August Kekule ^ succeeded
in preparing glycogen free from nitrogen and ash.

We shall later find that this skillful investigator, Bernard, not onl}- dis-
covered glycogen, but to him is also due the credit of clearl}^ recognizing
its biological significance.

Glycogen is closely related to starch not alone by acting as a reserve
carbohydrate, but also as regards its formation. It is, however, not iden-
tical with starch, but sharply distinct from it. In common with the other
members of this complicated group of carbohydrates, its empirical formula
is represented by (CgHioOs)^. It is a fine, white, amorphous powder.
We know absolutely nothing concerning its molecular weight.' It swells
in cold water and apparently dissolves, although the solution shows a
distinct opalescence. That an actual solution is not formed is shown by
the fact that the glycogen will not diffuse through a parchment membrane.
Furthermore, Gatin-Gruzewska ^ has recently shown that glycogen in
water behaves exactly like a colloid, migrating towards the anode. Gly-
cogen is de.xtrorotary. Its solutions are colored by iodine yellowish-
brown, reddish-brown to deep red according to the concentration. .\n
alkaline solution of copper oxide dissolves it, but the solvent is not
reduced.''

Quite like the other polysaccharides, glycogen is decomposed by
boiling with dilute mineral acids into its simplest component, which in
this case is exclusively grape-sugar. The breaking down of the comple.x

' Clamle Bernard: Lemons svir la Physiologie et la Pathologie du Sy.steine nervou.x,
vol. i, p. 467 (1857). See also Gazette inedicale, 28, III (1857).

' Pliarin. Zentrb. p. ;500 (1858).

' Z. Gatin-Gruzewska: Wanderung dcs Glykogens unter dem Einfhi.ss dcs elektrischen
Stromes. Pfliiger's .\roh. 103, 287 (1904).

« Pfliiger's .\rch. 103, 'JSJ (1904).

' Concerning its quantitative estimation, see E. F. W. Pfliiger, loc. cit. pp. 61
and 67, et seq.

46 LECTURE III.

molecule can take place, as with starch, step by step, and quite anal-
ogous products are obtained here. Diastatic ferments likewise attack
glycogen. Among the decomposition products obtained by hydrolysis,
dextrin and maltose have been identified with certainty. In its other
relations it is exactly similar to starch, and here as in the case of the
compounds of higher molecular weight formed from it (the dextrins, for
example) , we have no guarantee concerning their individuality, and sim-
ilarly we do not really know whether glycogen itself is a simple com-
pound or a mixture.

We do not yet know with sufficient certainty whether glycogen as such
is deposited in the tissues, or whether at least a part of it may not be
present in a combined state.

Glycogen is widely distributed in the animal kingdom, and is found in
all sorts of different tissues.' One of its chief sources is the liver, in which
it is deposited in the cell-substance. The nucleus is always free from it.
The amount present depends greatly upon the condition of nourishment
of the animal. The liver contains this polysaccharide even in its early
stages of development,^ although perhaps in small amounts. It is also
found in organs corresponding to the liver in many invertebrates, thus, in
crabs, mollusks, etc.

Detailed studies have been made concerning the distribution of glycogen
in the liver of the Gasteropoda. It was found that the content of glycogen
was dependent upon precisely the same conditions as with Vertebrata.
In the case of Limax and Helix the entire glycogen content could be made
disappear at the end of twenty to twenty-one days. After feeding, it
reappeared in the course of nine or ten hours. It is first deposited in the
connective tissue, and then in the epithelium of the liver. Starvation
alone causes it to disappear. With the gasteropods the liver is the only
place in which glycogen is deposited to any extent; in the other organs
the amount is hardly worthy of consideration.

Also in the lower organisms, other than mollusks and gasteropods,
glycogen is widely distributed. Bernard found it in the larvae of flies,
the grubs of many insects, in earth-worms and tape-worms, etc. Other
authors have mentioned its occurrence in Echinoderms, Holothuria,
Polyps, Sponges, etc.

Glycogen has likewise been identified in Protozoa (Vorticella, Opalina,

' For the microchemical detection of glycogen, see Dietrich Barfurth: "Verglei-
chende histochemische Untersuchvmgen iiber das Glykogen," Arch, mikro. Anat. 26,
259 (1895), and Edgar Gierke: " Das Glykogen in der Morphologic des Zellstoffwechsels,"
Habilib-Schrift, G. Fischer, Jena, 1905.

^ E. Pflijger: Ueber den Glykogenhalt der fotalen Leber, Pfliiger's Arch. 95, 19
(1901), and Glykogengehalt der fotalen Leber und die Jodreaktion des Glykogens,"
ibid. 102, 305 (1904).

CARBOHYDRATES.

47

Chilodon, Amojba, Rhizopoda) and also in fungi.' Clautrian,^ as well as
Harden and Young,' has carefully determined and studied the amount of
glycogen in yeast.

The identification of the glycogen has in many cases not been perfect,
and it is an open question as to whether some of the supposed glycogen has
not been an entirely different substance. At all events, these substances
belong, according to their biological relations, to the glycogen or starch
group, or, as we may say, to the group of reserve-carbohydrates.''

In the vertebrates the muscles also serve to store glycogen. The various
muscles contain different amounts, as shown by the following table: ^

Dog I .
Dog II .
Dog III
Rabbit I

Biceps brachii . .
Quadriceps femoris
Biceps brachii . .
Quadriceps femoris ,
Dorsal musculature
.\dductors posterior
Dorsal musculature
Adductors posterior

Per cent.

jO.17

iO.53

(0.25
(0.32

J 0.135
(0.077

J 0.417

? 0.444

It is found not only in striated but also in smooth muscle and in the
muscle fibrils. The glycogen content in the muscles is dependent upon
the condition of nourishment. We shall soon see that the glycogen of
muscles has a particular function, and stands in direct relation to the
performance of work by the musculature. Even in invertebrates, glycogen
is not lacking in the muscular apparatus, and performs the same function.

Glycogen occurs furthermore, in the pancreas, in the small glands of
the digestive apparatus, the lungs, kidneys, sexual glands, brain, epithe-
lium, connective-tLssue, and blood- " and lymph vessels.

B. Schondorff ' has determined the amount of glycogen in different

' Errera: "Das Epiplasma der Ascomyceten und das Glykogen der Pflanzen," Briissel
(1882), and Corapt. rend. 101, 253 (1885).

' Clautrian: "Chemische Untersuchungen iiber Glykogen," Mem. couronn. Acad.
Roy. Belg. p. 53 (1895).

» Trans. Chem. Soc. 81 (1902).

* Concerning the occurrence of glycogen under pathological conditions, see O.
Lubai^ch in O. Lubarsch und R. Ostertag: Ergebnisse, 1, Jahr. 2, 166 (1895).

' August Cramer: Z. Biol. 24, 78 (1888).

' It is a much disputed question whether blood-plasma itself contains glycogen, or
whether the glycogen in blood is to be traced merely to that of the white blood-corpuscles.
It looks as if glycogen might be present in the piasra^, though ordinarily its presence
is limited to the leucocytes.

' Pfliiger's Arch. 99!^ 191 (1903).

48

LECTURE III.

organs of dogs which were well fed with carbohydrates and meat shortly
before their death. The following table gives a summary of the results
obtained:

PER CENT OF GLYCOGEN IN THE ORGANS.

Blood
Liver
Muscle .
Bone
Viscera
Skin .
Heart
Brain

Dog I.

.005

II.

III.

IV.

V.

VI.

0.002

0.0061

4.35

7.60

18.69

17.10

16.38

9.89

0.72

0.88

2.54

3.23

3.72

2.53

0.18

0 39

1.00

1.31

1.76

0.97

0.03

0.08

1 47

1 .51

1.72-

1.01

0.38

0 20

0 73

0,84

1 60

0.92

0.12

0 10

0,58

0.72

1 .21

0,49

0.04

0-23

0.27

0.23

0.20

0.25

100 grams of glycogen in the body are distributed in the different parts of the
dog as follows:

Dog I.

II.

III.

IV.

V.

VI.

VII.

Average.

Blood . . .

0.04

0.01

0.001

0,015

Liver .

20.09

26.37

53.54

56.74

38.53

21 .95

48.54

37.97

Muscle .

62.55

58.31

31.22

29.00

38.93

53.76

35,83

44,23

Bone

5,36

10.32

6.81

7.29

12.88

11.30

10 77

9,25

Viscera

0,38

1.10

5.21

4.31

5.32

7,30

3,03

3.81

Skin . .

11.38

3.76

3 00

2.48

4 01

5,38

1,42

4.49

Heart .

0.17

0.08

0.14

0 12

0.28

0,18

0,19

0.17

Brain

0.04

0.06

0.07

0.05

0.05

0.13

0.23

0.09

It is evident from the above table that the amomit of glycogen present
in the different organs varies greatly. At all events, it is never possible to
draw conclusions concerning the amount of glycogen present in any given
organ from a knowledge of the amount containeil in another organ or
even in the whole body.

Besides the carbohydrates which have been mentioned up to this point,
there are quite a number of other compounds belonging to the group of
polysaccharides which have been observed in blood, in milk, and es-
pecially in urine. They have been designated partly as animal gums,* and
partly as dextrin-like substances,^ etc. The last-mentioned are found in
large quantities in the urine of diabetics, although it- is quite possible that
such products may be present to some extent even in normal urine, because
boiling it with mineral acids causes the formation of humin substances,

' H. A. Landwehr: Zent. med. Wissensch. 21, 369 (1885). See also K. Baisch: Z.
physiol. Chem. 18, 193 (1.S94); 19, 339 (1895); and 20, 249 (1895).

' Cf. K. V. Alfthan: Helingfors. Osakeyhtio Weilin und Goos Aktiebolag. 1904.

CARBOHYDRATES. 49

which points to the presence of carbohydrates.' Our knowledge concern-
ing thase products is still indefinite, and the same may be said concerning
their biological significance. It seems possible that in the case of these
complicated carbohydrates in urine we have to do with products which
have escaped a complete breaking down.

At this place we may mention the arid recently oljserved by P. A.
Levene ■ in the preparation of nucleic acid from the spleen, which of itself
has no reducing power, but acquires it after being boiled with acids.
Levene called it glucothionic acid, and regards it as a sulphuric acid
ester. It is not yet determined what the nature of the carbohydrate
component is. John A. JIandcl and P. A. Levene ' succeeded in obtaining
this acid also from the kidneys, liver, pancreas, and milk glands, although
only in very small amounts. Apparently such sulphuric acid compounds
of carbohydrate-like substances are quite widely distributed in the organ-
ism. Nothing is definitely known as to the relation that chondroitin-sul-
phuric acid * (prepared from cartilage and amyloid) bears to this group,
and we are equally ignorant concerning the significance of these products.

Our knowledge concerning jecorin, first described by Drechsel ^ and
found by him in the liver of a horse and later in that of a dolphin, and
finall}' by Baldi ° in the same organ and spleen of other animals, in the
muscles and blood of the horse and in the human brain, is still very indef-
inite. Its constitution is unknown, but it contains sulphur, phosphorus,
and a carbohydrate complex which Manasse ^ states to be glucose.^ It is
probably not a chemical individual, but rather a mixture of quite dif-
ferent products. At present there is not much known concerning its
significance."

' Cf. Emil Abderhalden and Fritz Pregl: Z. physiol. Chem. 46, 19 (1905).

' Z. physiol. Chem. 37, 400 (190.3).

' Z. physiol. Chem. 45, 386 (1905).

« Carl Th. MOrner: Z. physiol. Chem. 20, .357 (1895). See also Skand. Arch. Physiol.
1, 210 (1899); Z. physiol. Chem. 23, 311 (1S97). N. P. Krawkow: Arch. exp. Path.
Pharm. 40, 195 (1898). R. Oddi: ibid. 33, 37G (1894). O. Schmiedeberg: ibid. 28, .ioo
(1891). A. Orgler and C. Xeuberg: Z. physiol. Chem. 37, 407 (1903).

' E. Drechsel: Ber. sachs Gesell. Wis.sensch. 1886, p. 44, and " Beit rage zur
Chemie emiger Seetiere," Z. Biol. 33, 85 (1890).

• Baldi: Arch. Physiol. 1887, Suppl. p. 100.

' Z. physiol. Chem. 20, 478 (1S95).

' B. Biiig. Skand: Arch. Physiol. 9, IGO (1900).

» Pee also J. Meinertz: Z. physiol. Chem. 46, 376 (1905), and M. Siegfried u. II. .Mark:
i6iV/. 46, 492 (1905).

LECTURE IV.

CARBOHYDRATES.

III.

Metaboli.sm of Carbohydrates in Plant and .\nimal Organisms.

Formerly the biology of plants occupied a sharply distinct field from
that of the animal organism. The two kingdoms were believed to be
opposed to one another with regard to the transformations of energy and
of force taking place within each. Plants were alone held to be capable of
building up organic substances, i.e., to be capable of effecting syntheses.
The animal system, on the other hand, served to break down such sub-
stances. In this way the animal and vegetable worlds acted in conjunction
and formed a large unit. However, the more scientists penetrated into
the intricacies of vegetable and animal metabolism, and in proportion to
the comparative studies made, the more it became evident that there was
no sharp line to be drawn between these two fields. When Wohler in 1824
discovered that benzoic acid introduced into the animal body was not
consumed nor eliminated as such, but was to be found in the urine com-
bined with glycocoll in the form of hippuric acid, the path was broken,
and for the first time a synthetical process was recognized as taking place
in the animal organism.

In the following period, as we shall see, a large number of such syntheses
were discovered as taking place in the organism of animals, and to-day
there is no longer any doubt but that synthetical processes play an
important part therein. To be sure, in importance, and, as far as we know,
in variety also, they are far in the background as compared to the syntheses
in plant organisms. On the other hand, plants utilize oxygen and produce
carbon dioxide from more complicated compounds ; in other words, they
break down substances. In this way physiology has given a new and pow-
erful support to the well-known common morphological outlines of the two
kingdoms, so that to some extent the two fields have been placed upon a
common basis although each retains a certain degree of independence.

Nothing supported the old conception of the sharp distinction between
the synthetical processes of plants on the one hand, and the catabolic pro-
cesses taking place in animal organisms on the other hand more than the

50

CARBOHYDRATES. 51

formation of carbohydrates in plants.' No synthesis is more wonderful
than this. It determines the whole metabolism of the plant; it forms
the support upon which rests the whole development or extinction not
only of plants but of the animal world. By means of it the great mass of
carbon which, in the form of carbon dioxide, is apparently withdrawn
from metabolism as the final product in the combustion of organic sub-
stances, is carried back to it ; thus the sugar synthesis, or what is commonly
spoken of as the a.ssimilation of carbonic acid by the organs of the plant
containing chlorophyll, forms an important stage in the carbon cycle.
The great deposits of coal, and the rocks which underlie the surface of
the earth in vast layers, and are to a considerable extent composed of
carbonates (principally of calcium and magnesium), belong to this cycle.
The coal is formed from plant residue which formerly thrived on the
carbon dioxitle of the air; and by burning coal, carbon dioxide is again
formed, so that the cycle can again take place. Carbon dioxide found
combined with bases such as lime and magnesia likewise originated from
the atmosphere. It is temporarily removed from the cycle only to return
to it when, for example, it is replaced by the action of another acid, such
as silicic acid. Oxygen, for the greater part, also makes the cycle with
carbon.

The only important source of the carbon contained in plants is, in fact,
as Ingenhousz - and then Theodor de Saussure ^ first showed, the carbon
dioxide of the air. It is true that it has been observed that roots can
take up carbonic acid from carbonates and bicarbonates in the soil, but
the amount thus available is very slight. Carbon dioxide is taken up
for the most part through the stomata (breathing-pores) of the leaves.
The a.ssimilation depends within certain limits upon the amount of car-
bon dioxide in the atmosphere, the temperature of the leaf, and the
intensity of the illumination.^ For every temperature there is a definite
amount of carbon dioxide assimilation; in general, the optimum lies
between 20° and 30° C.

In spite of numerous studies, it is still a problem as to what is first
formed from the carbon dioxide. .\t present we understand merely the

' Concerning the assimilation of carbonic acid, consult the text-books on botany, e.g.,
W. PfeEFer's " Pflanzenphysiologie, Ein Handbuch der Lehre vora Stoffwechsel und
Kraftwecheel in der Pflanze," pubHshed by Engelmann in Leipsic. — Friedrich Czapek:
"Biochemie der Pflanzen," [niblished by P'ischer in Jena.

' Ingenhousz: " Exporimonts upon Vegetables," London, 1779, and "Essays on the
Foods of Plants and the Renovation of Soils " (1790).

' T. de Saussure: "Recherches chimiques sur la v^g^tation," Paris, 1804. —
Edmund O. v. Lippmann: "DieChemie der Zuckerarten." — T. Sachs: "Geschichte
des Botanik." — A.Hansen: "Geschichte der .^.ssimilation."

* F. Frost Blackman and Gabrielle L. C. Matthaei: Proc. Roy. Soc. London, 76, 402
(1905).

52 LECTURE IV.

outward conditions under which the carbonic acid assimilation takes
place. We know that cells containing chlorophyll are absolutely indis-
pensable to the process. In order to disturb the condition of equilibrium
in the carbon dioxide molecule and in those of the water required to form
the assimilation products of carbon dioxide — in each of these compounds
the affinity of carbon and hydrogen for oxygen is completely saturated —
energy is necessary. The plant cells perform work in transforming
kinetic energy into potential energy. It has been known for a long time,
and proved experimentally, that cells containing chlorophyll are not of
themselves capable of assimilating carbonic acid. The process takes
place only by the aid of light. The light viljrations of the ether furnish
the energy. All the rays of white light are not active in this respect.
In fact, the so-called chemical, or actinic rays, of the ultra-violet and
similarly the peculiar heat rays of the infra-red part of the spectrum
have little or no power of furnishing the cells with the energy necessary
for assimilation. The most active rays are the red, orange, and yellow.'
These relations have been established by the work of Engelmann.^
The essential moment, therefore, in the formation of organic substances
in the plant cell is the transformation of radiant energy into chemical
energy, a process which, as far as we know, takes place exclusively in
cells containing chlorophjdl. Chlorophyll takes part, thereby, in an
important phase in the energy cycle. By its help, kinetic energy is trans-
formed into potential energy, and when later the plant is eaten by the
animal, this is eventually changed back into kinetic energy. Yet even in
the vegetable world the last-mentioned process plays an important part,
for we know of whole groups of plants which are not able to assimilate
carbon dioxide themselves; these are the parasites which contain no chloro-
phyll, and, as regards their metabolism, they are closely related to the
animal organism; while on the other hand, we have species belonging to
the animal kingdom (vorticella, certain Flagellata, planarians, hydra, etc.),
which assimilate carbonic acid, and set free oxygen, thus imitating plants
in their metabolism. It has been found that the assimilation of carbonic
acid in such cases is also due to the presence of the same agent, chlorophyll,

' The maximum assimilation for tlie bluish-green, fresh-water algs and the red sea-
algas is effected by rays in other pai'ts of the sun's spectrum. Engelmann has shown
(loc. cit.) that the light rays act more strongly in proportion as they are absorbed by
the color. In general, light complementary to the color of the plant is most active upon
assimilation. Spectroscopic analysis shows that of light passing through different
depths of water, the red rays are strongly absorbed, while the green and bluish-groen
are less so. This explains why at greater depths of water, the red and yellow forms
prevail rather than those which are blue or bluish-green in color. Cf. W. Engelmann:
Arch. Physiol. Suppl. 1902, p. 3.3.3. Gaidukow: ibid. p. 214.

' Th. W. Engelmann: Bot. Ztg. 1882, 419; 1883, 1, 1884, SO; 1886, 64; 1887, 393,
Pfliiger's Arch. 25, 285 (1881); ibid. 26, 537 (1887); 27, 485 (1882); 30, 95 (1883).

CARBOHYDRATES. 53

which is present in plants; and in fact, the chlorophyll is not deposited free
in the tissues of these animals, but the chlorophyll-bearers are those algae-
which exist together with the animal cell and have a commensal existence.
This sort of parasitism is met with very frequently, for instance in
lichens,' which are composed of fungi and algae. Again, we find the same
sort of symbiosis in higher organisms. Undoubtedly, for example, the
bacteria found in the alimentary canal illustrate this relation; they serve,
as we shall see later on, to convert carbohydrates (cellulose, etc.) into a
form capable of alisorption.

By means of carbonic acid alone, the cell cannot build up organic sub-
stances; hydrogen, which forms an integral component of all the com-
plicated compounds of the carbohydrate series which the plant organism
produces, is lacking. -Hydrogen is obtained by the plant chiefly from
water in the soil. Herein the plant finds another source of oxygen. The
way in which the plant cells utilize, by the help of chlorophyD and the
sun's rays, this carbonic acid and water, or, in other words, the first
products formed, is still a matter of conjecture. We know merely that
all syntheses start with a reduction process; oxygen is set free. In fact,
we can follow the assimilation of carbonic acid either by determining the
loss of carbonic acid in a gas mixture, or by determining the oxygen
evolved. The latter can be observed directly by immersing the parts of
a plant in water. Engelmann ^ ingeniously made use of bacteria as a
reagent for oxygen. If, for example, a thread of alga and some aerobic
bacteria are placed under an air-tight cover-glass, it will be noticed at
first that the latter are very lively. If, now, the preparation is kept in the
dark the motion of these bacteria will cease altogether after a time, show-
ing that all the oxygen has been consumed. Then, on bringing the prep-
aration to the light, the bacteria begin to become most active because the
thread of algae has begun to assimilate carbon dioxide and water with
elimination of oxj-^gen. By means of this very sensitive test as little aa
one l)illionth part of a milligram of oxj'gen may be detected. Engelmann,
as already stated, has by means of this method studied the different parts
of the solar spectrum, testing it with regard to its effect upon plant assim-
ilation. Beyerinck ' later in a similar way made use of the luminescence
of certain bacteria, which also depends upon the presence of free oxygen,
for the detection of carbonic-acid a-ssimilation.

With regard to the synthetical products first formed by the plant,

' Schwendener: Xagelis Beitrage z. wissensch. Botanik H. 2, 3, and 4, Leipsic, 1800-
ISGS. — de Bary: " Die Erscheinung der Symbiose," Strassburg, 1S79. O. Hertwig:
"Die Syrabiose oder das Genossensehaftsleben in Tierreich," Jena, 1883.

' Bot. Ztg. loc. cit.

^ Ibid. (1890) 744. See als;o Hans Molisch: "Die Lichtenwicklung in den Pflan-
zen, Xaturwissen.schaftliche Rundschau, 20, 505 (1905).

54 LECTURE IV.

different conjectures have been made, and the same is true concerning
the action of chlorophyll. Some conceive it to have the action of a fer-
ment,' others consider it as closely related to the first assimilation product
assuming that it combines with the carbonic acid.

As long as we know practically nothing concerning the formation of
chlorophyll, and still less about the plasma of the plant cell itself, and as
long as we are unable to isolate satisfactorily any of the first products of
the assimilation, the discussion of all such possibilities has only a relative
value. For the present we can restrict ourselves to those hypotheses
"which seem plausible from a chemical standpoint. At the same time it
does not follow necessarily that the plant organism carries out its syntheses
in such a way, nor that the intermediate products which we assume are
actually formed in the living plant cell. Above all, the impression should
not be left that the assimilation of carbonic acid can take place only in one
direction. It is indeed po.ssible, and in fact probable, that other primary
products of assimilation exist. It is undoubtedly certain that one of the
first products of the assimilation is a carbohydrate, whether grape-sugar,
starch, or perhaps a simpler sugar with fewer than six carbon atoms in the
molecule. At all events, — and this is of greatest importance for under-
standing the whole metabolism of plants, and indirectly that of the animal
organisms, — the carbohydrates stand at the center of all the synthetical
processes taking place in the plant organism. We shall see later on that
the fats, the albumins, and many other highly complicated compounds,
evidently originate from carbohydrates, whether by constructive or
destructive processes, or both acting together.

In discussing the first synthesis produced in the plant cell we meet with
a highly important problem which we have already briefly touched upon.^
All the carbon compounds which are either directly or indirectly produced
by animal or plant organisms, are optically active; that is to say, they
possess at least one asymmetric carbon atom. As we have already seen,
most of these compounds are found in nature, only in the optically-
active state. Now, the plant cell in carbon dioxide receives a carbon
compound, which does not contain an asymmetric carbon atom. The

' Jean Friedel (Compt. rend. 132, 1138 (1901)) has tried to support this view by-
experiments. He brought- together the glycerol extract from fresh leaves with finely
powdered leaves which had been rapidly and carefully dried, and found oxygen was
evolved by the action of light, and carbonic acid taken up. The experiments, however,
are not conclusive, and have not been corroborated. Cf. M. Harroy: Compt. rend. 138,
890 (1901).— R. O. Herzog: Z, physiol. Chem. 35, 459 (1902). Again, Bach and Chodat
have published a great deal of work summarized in Biochem. Zentrbl. 1, 11, 417, and
I, 12, 457 (1903).

The relations are so complicated that it is hard to draw a conclusion at the present
time concerning the work of these scientists.

2 Cf. p. 15.

' CARBOHYDRATES. 55

beginning of all the asymmetrj- in the organic world is centered in the
assimilation of the carbon dioxide, for evidently it is here in the plant
cell that the first asymmetric synthesis is carried out which continues
constantly in the production of only asymmetric compounds.' It is
indeed possible that chlorophyll, which is itself a.symmetrically consti-
tuted, plays an important part in this asymmetric synthesis. The first
appearance of asymmetry is, however, unexplained. Its existence evi-
dently coincides with the formation of the first cell. It is possible, on
the other hand, that the plant cell produces first of all an inactive com-
pound, for example, an inactive sugar, and that asymmetry first develops
by the cleavage of this compound.- The animal organism receives in turn
with its food, this asymmetry of its body-substance from the plant
world, partly directly, as in the case of the herbivora, and partly
indirectly, a.s in the case of the carnivora. We shall find, moreover,
that these relations are so suited to the animal organism that it some-
times directly disintegrates racemic compounds, and in many cases utilizes
only one component and discards the other unchanged.

The fii-st observed assimilation product of the carbon dioxide and water
was starch, easily recognized by means of the iodine reaction. For a long
time it was held to be the primary product of a.ssimilation. Gradually it
became a recognized fact that starch is a product formed secondarily from
more simple components, and little by little the opinion gained ground
that rf-glucose was to be regarded as the primary product of assimilation.
This assumption was supported by the discovery that leaves which have
been preserved in the dark are capable of forming starch directly if solu-
tions containing hexoses are placed upon them. In general, the following
compounds are assimilated: — d-glucose, rf-mannose, d-galactose, and
d-fructose. Other similar compounds may be utilized; thus, mannitol

' Cf. Emil Fischer: "Die Chemie dor Kolilehydratc uml ihre Bedeutung fur die
Physiologie."

' Cf. A. Byk: Z. physiol. Chem. 49, G41 (1904). Byk seeks to trace the formation
of asymmetry back to circularly-polarized light which maybe produced by the reflection
of plane-polarized daylight by the surface of the sea. The revolution of the plane of
polarization by the magnetism of the earth makes it impossible for equal amounts of
the two forms of light to exist at any point on the earth, or upon the whole surface
of the earth, or for any considerable period. It is indeed possible that such may be
the explanation of the a.symnietrj- of the first cells, Viut it is scarcely probable that it
accounts for the continuous production of a.symmetric molecules, for it is unreasonable
to assume that one and the same kind of light is permanently in excess at one and the
same point on the earth's surface: and it would seem likely, furthermore, that compo-
nents of unlike optical products would be formed in different localities, .\gain. Byk s
proof that by means of circularly-polarized light racemic compounds can be split off is
not perfectly satisfactory. Furthermore, chlorophyll itself has optical properties. It
undoubtedly changes the sun's rays of short wave lengths, which have no effect upon
the assimilation, into active, longer wave lengths.

56 LECTURE IV.

by leaves of the Oleacese, dulcite by those of the Evonymus; and interest-
ingly enough it has been found in the case of all the plants examined that
the assimilation takes place with those carbohydrates which they contain
normally as reserve-substances.

The fact that the volume of carbon dioxide absorbed is equal to that of
the oxygen evolved ' has been cited to prove that a carbohydrate must be
the first assimilation product.'^ Again, the relations of the heat effect
observed coincides very satisfactorily with this assumption. On the other
hand, it must be remembered that it has not been possible to carry out
exact measurements in this direction. Side by side with the assimilation
of carbon dioxide there is a constant absorption of oxygen and production
of carbon dioxide. Both processes stand in a certain relation to one
another, although the nature of this has never been determined. It has
been repeatedly asserted that oils and fats may appear as the first pro-
ducts of assimilation. Inclusions of oil have been observed frequently
in the chromatophores of certain plants; for example, in the Musaceee,
Cactaceae, algae, and especially the Vaucheria.^ By the decomposition of
the fat into glycerol and fatty acids, and the simultaneous partial reduc-
tion or oxidation of these cleavage products, sugars could be formed from
the glycerol, and vegetable acids from the fatty acids. It was soon evi-
dent, however, that these fats and oils were not primary products of
assimilation, but rather reserve-substances, the formation of which can
l)e readily traced back to carbohydrates. Equally untenable proved
Liebig's * hypothesis that the vegetable acids were formed as the first
assimilation products from which the carbohydrates were obtained
secondarily. At present, there are no known observations which are
contrary to the assumption that the carbohydrates represent the entrance
of the carbon from carbon dioxide into the general metabolism of the plant.

It is now a question as to how we shall explain this sjmthesis of carbo-
hydrates — rf-glucose, for example — from carbon dioxide and water. We
have already mentioned ^ one hypothesis, namelj^, the assumption of
Baeyer ° that carbon dioxide by reduction is changed into formaldehyde,
and by the condensation of the latter a sugar is formed:

CO2 4- H20->HCH0 + O2.

Baeyer originally assumed that the carbon dioxide absorbed was first

' Boussingault: Compt. rend. 53, 862 (1861), and Holle: Flora, 118 (1877).
= This might take place as follows: 6 CO, + 6 H,0 = CsH,jO„ + 6 O,.
' Paul Fleissig: " lleber die physiologische Bedeutung der Olartigen Einschliisse in
der Vaucheria," Inaug. Diss. Basel. 1900.
* J. Liebig: Ann. 46, 58 and 66 (1843).
' Cf. p. 15.
« Ber. 3, 03 (1870).

CARBOHYDRATES. 57

changed into carbon monoxide and oxj'gen. The former was behaved to
combine with chlorophyll in much the same way as it does with haemo-
globin.

Bach ' has recently modified somewhat this suggestion of Baeyer. Accord-
ing to him, the carbonic acid is first changed into percarl)onic acid, water,
and carbon. The percarbonic acid is then decomposed into carbon
dioxide and hydrogen peroxide, while the latter forms with carbon and
water the formaldehyde:

3 H2CO3 = 2 H2CO4 + HoO + C,
2 H2CO4 = 2 CO2 + 2 H2O2 = 2 CO2 + 2 H2O -F O2,
H2O + C = HCHO.

Naturall}', a great many attempts have been made to isolate formalde-
hyde or related compounds from plants, especially from the green leaves.
This has not been accomplished, however, tipTcTtlie present time in a_satis-
factory way.^ On the other hand, nothing has been shown that is con- ^
trary to the assumption that formaldehyde does actually represent the(aft<-
first intermediate product, for it is perfectly possible that the amount of . ^, ±^
aldehyde that is constantly being formed is so extremely small that it ' v,___-,^
condenses so rapidly that it escapes detection. It has been brought
forward in support of Baeyer's theory that certain plants show a con-
siderable resistance towards formaldehyde. Thus Treboux ^ found that
Elndea canadensis will stand a 0.001 per cent solution. Alg£e and young
plants of Sinapis alba are said to be strikingly resistant towards formal-
dehyde.*

We saw in considering the artificial synthesis of carbohydrates that
it was possilile to form a sugar very easily from formaldehyde, and we
can readily understand how, according to the number of molecules of
formaldehyde entering into the reaction, sugars containing a different
number of carbon atoms in the molecule may be obtained. It is, how-
ever, scarcely probable that such syntheses take place in this simple way.
Thus, for example, according to all that we know at present, it is hardly to
be expected that pentoses are formed directly as one of the first products
of the condensation of formaldehyde. Apparently it is much more likely
that such sugars are formed by the breaking down of higher sugars
especially the hexoses. Such a process is likewise easy to represent, as, in

' Arch. sri. phys, nat. Ocn. 5, 101 (ISOSV

^ H. Euler: Ber. 37, .3111 dOOO. Hans and .\strid Euler: Arkiv fiir Kemi. 1, 347
nnCMI : Ber. 39, 39 (190G\ and ibid. 39, 45 (1906). W. Loeb: Z. Elektrochem. 11, 745
(1905"!.

' Trt'houx: Flora, 73 (1903).

* R. Boiiillac: Compt. rend. 135, 13G9 (1902). Bouillac and Giustiniani: ibid. 136,
1155 (1903).

58 LECTURE IV.

fact, 0. Ruff has shown.' He prepared d-gluconic acid by the oxida-
tion of d-glucose; its calcium salt he allowed to stand in the sunlight in
the presence of ferric acetate, or he treated it with h\-ilrogen peroxide
and thus obtained d-arabinose.

CHO CO.OH CHO

HCOH

Hc;oH

1

HOCH

HOCH

1

HOCH

' HCOH

1

HCOH

HCOH

HCOH

HCOH

1

HCOH

1

CH2OH

rf-glucose

CH2OH
rf-gluconic acid

CHoOH

rf-arabinose

At all events, — and this is of great importance, — we can explain
chemically without difficulty the formation of the different members of
the carbohydrate series from the hexoses, especially c?-glucose.

Emil Fischer - has suggested that the glycerose discovered by him is
perhaps the first assimilation product of carbonic acid by the plant cells
containing chlorophyll. By the combination of two molecules of this, it
is easy to understand how hexoses may be formed. Again, glycerose maj'
be of great importance in other relations. We shall mention here merely
the fact that it is closely related to glycerol, which is one of the components
of the fats; and on the other hand, that from this point of the carbon dioxide
assimilation the synthesis of albumin may start. Glycerose, as we shall
show more fully later on, stands in close relation to certain of the decom-
position products of albumin, namely alanine, serine, and cystine. Naturally
it does not necessarily follow that either the formation of the fats or that
of the proteins inust begin with this hypothetical product of assimilation.
It is indeed possible that glycerose appears merely as one of the decom-
position products of c?-glucose or starch, and then is used for syntheses
by the plant cell.

All of these possibilities have been brought forward in order to bring
the ground-plan of the whole carbon assimilation at least within the realms
of our understanding, and to show the far-reaching value that purely
chemical investigations, especially those of Emil Fischer, have upon the
science of biology; for it is by this means only that the most important
classes of substances — carbohydrates, fats and proteins — have been
traced back to a common source. The consideration of these relations is

' Ber. 31, 1573 (1898); 32, 550 (1899). Otto Ruff and Ollendorf: ibid. 33, 1798
(1900). Cf. A. Wohl: ibid. 26, 730 (1893) ; 33, 3066 (1899).
' Ber. 23, 2138 (1890).

CARBOHYDRATES. 59

of great importance; and a detailed discussion is justifiable, furthermore,
because it is very probable that also in the animal organism chemical
changes take place by means of which a substance belonging to one class
is changed into a compound of another, as, for example, a fat into a car-
bohydrate or conversely.

The animal organism is, in its entire existence, dependent upon this
carbonic acid assimilation by the plants, for in this way it obtains all the
organic compounds of complicated structure. Energy originating in the
sun is thus obtained by the animal in the form of potential energy, from
which the animal derives its kinetic energy and abilitj' to perform work.
The assimilation by the plant not onl}- serves to furnish organic material
for the animal organism, but to a certain extent it furnishes the oxygen
which it requires for obtaining the kinetic energy again by combustion;
the a.ssimilation process being one of reduction in which oxygen is con-
stantly being evolved. The oxygen in this way returns to the general
cycle of the elements. This oxygen, after taking part in the metaliolisra
of the animal, escapes chiefly as carbon dioxide and water, both of which are
again utilized by the plant for the formation of other organic substances.

Let us turn now to those carbohydrates which are most important as
food for the animal organism, namely, grape-sugar, cane-sugar, and starch.
From the last two compounds the animal obtains all the carbohydrates
that it needs. Let us follow these sugars on their way through the ali-
mentary canal to their absorption and final assimilation. As an example,
we shall choose starch, because it is here that the relations are the most
complicated, and we shall be able to treat of the behavior of the
simpler sugars in connection with the separate phases in the breaking
down of the starch.

First of all starch — or, strictly speaking, the food containing it — ■
is ground up with the saliva by the act of chewing. The saliva con-
tains a diastatic ferment, ptyalin,' which converts the starch into dextrins
and finallv largelv into maltose." The latter is inverted by means of a

' Ptyalin is not found in the saliva of all animals, e.g., that of the camivora. It
would be well to drop the name ptyalin a-s it tends to give one the impression that
there is only one ferment found in the saliva. At present we know only of its
action, which coincides with that of various other ferments found in the animal and
vegetable kingdoms. It is better, therefore, to speak of a diastatic or amylolytic fer-
ment. We would be justified in using a special name for the ferment of unknown com-
position only when it has an unusual action; thus, for example, if the diastase in saliva
led to different cleavage-products of starch than do the ferments of other origin.

' The earlier assumption, that glucose is formed directly, has been shown to be false.
Cf. J. Seegen: Zent. med. Wissen.sch. 14, 849 (1S7G), and Pfliiger's Archiv. 19. 106
(1879). Otto Na-sse: ibid. 14, 47:{ (1877). Musculus and v. Mering : Z. physiol.
Chem. 1, 39,5 (1S77-7S). Thi.l. 2, 403 (1878-79); il)id. 4, 9.3 flS.SO). von Mering: ihid.
5, 185 nSSl). Brown and Heron; Ann. 199, 165 (1879); 204, 228 (1880). Kiilz and
Vogel: Z. Biol. 31, 108 (1895).

60 LECTURE IV.

special ferment, called glucase (or maltase)} This last action plays a
subordinate part in the total action of the saliva. The transformation
of the starch past the dextrin stage into maltose does not take place so
simply as might seem possible. A great many intermediate products have
been described. At present there is no reason for going into the details here,
partly because we are not sure whether some of these products are single
substances, or mixtures of several constituents.

The human saliva, or rather the amylolytic ferment contained in it,
does not attack raw starch to any extent. In almost every case the
starch has already undergone a process of change which greatly facil-
itates the action of the diastase upon it. In most cases the starch has
been cooked, which swells the grains. The fact that this is favorable to
the action of the diastase can be easily shown by the following experiment:
In one test-tube a few grains of ordinary starch are mixed with saliva,
while in another the same saliva is allowed to act for an equal length of
time upon starch paste. If at the end of a certain length of time iodine is
added to test for unchanged starch, a much stronger coloration will be
obtained in the first tube than in the second. If, on the other hand, we
test for the sugar ^ formed, we shall this time obtain a better test in the
other test-tube.

Leaving the mouth, the starch, together with some of its transformation
products, all intimately mixed with the saliva, reaches the stomach.
For a long time it was believed that the action of the diastase quickly
stopped here on account of the acid reaction of the contents of the stomach.
Free hydrochloric acid is especially unfavorable to the action of diastase;
as little as 0.03 per cent prevents it from changing starch into sugar.
Recent experiments ^ have shown, however, that the food reaching the
stomach is not immediately mixed with the gastric juice as was formerly
assumed, but on the contrary lies out of contact with this for some time.
In the stomach itself there has not been found any agent capable of con-
verting carbohydrates into sugar,** but, on the other hand, a not incon-
siderable absorption of the simple sugar formed is known to take place
here.

The main digestion of carbohydrates is effected, however, in the intes-
tine by the action of a diastase from the pancreas. By means of it the
starch — which for the greater part is still unchanged, or at least only very

' M. C. Tebb: J. Physiol. 15, 421 (1894). Hamlnirger: Pfluger's .'^rchiv. 60, 543
(1895).

' Naturally, the saliva, starch, and starch paste should be e.xamined for sugar at the
start.

3 p. Grijtzner: Pfluger's Arch. 106, 463 (1905).

* According to H. Friedenthal, the stomaeh juices of a dog contain a ferment which
acts strongly in acid solutions. Archiv. (Anat. und) Physiol. 1899, Suppl. 383.

CARBOHYDRATES. 61

incompletely broken down — is now acted upon completely, and in fact
intermediate products are formed which are similar to, if not identical
with, those produced by the saliva. The cleavage produced by the pan-
creatic diastase is believed to yield finally only maltose. This last, by the
action of a particular ferment, glucasc (also called maltase), is eventually
decomposed into molecules of rf-glucose. Here in the intestine the breaking
down of cane-sugar also takes place for the most part. By means of the
above-mentioned ferments, or at least by similar ones,' it is similarly
inverted into its two components, dextrose and Icevulose (d-glucose and
d-fructose). In this way the carbohydrates in the food are prepared
for absorption, which sets in as fast as the breaking down of the
carbohydrates takes place.

We must now touch upon the question as to whether the more com-
plicated carbohydrates, such as the dextrins, for example, are capable
of direct absorption. Under normal conditions these substances are not
taken up by the intestine, or at least such products are not met with
beyond the intestines in the assimilatory tracts.^ Cane-sugar and mal-
tose can be absorbed directly. They are taken up more slowly than the
simpler sugars, and in every case tliey must suffer cleavage before they
are turned over to the blood; for if cane-sugar, avoiding the intestinal
canal, is introduced directly into the blood, it suffers no further change,
but is eliminated as such in the urine.'

The most important result of the successive changes which take place in
the intestinal canal as regards the carbohydrates which we have studied
up to this point, is the tendency to form by the action of a definite ferment
the simplest building material, especially the hexoses, thus giving to the
system a uniform material from which it can construct the substances of
which the body is composed. It is evident from what has been said that
the significance of the alimentary canal and of all the different organs
connected with it does not consist solely in transforming the non-diffusible
substances, which cannot be absorbed, such as starch, into products
which are diffusible. Its task stretches far beyond this single action.*
The molecules which are naturally foreign to the animal organism
are destroyed and converted into a homogeneous indifferent material

' It is probable that the same ferment does not act upon both maltose and cane-
sugar. Cane-sugar is not split up beyond the intcstino, while maltose appears as a
product from glycogen and is inverted.

' Von Mering (.A.rch. f. anat. und Physiol. 1877, 379, 41.3) has found substances
similar to dextrin in the blood of the portal vein after a diet very rich in carbo-
hydrates. It has not been shown whether this is a normal occurrence.

= Cf. Fritz Voit: Deut. Arch. klin. Med. 58, 523 (ISO?). Ernst Weinland: Z. Biol.
47, 279 (1905).

* Cf. Emil Abderhalden: Zentrb. Stoffwechs. Verdauungskrankheit, 5, N'o. 24,
647 (1904).

62 LECTURE IV.

from which the organism is able to build up its own individual
carbohydrates.

Before we attempt to follow the simple sugars on their way to the
organs, or, in other words, to study the course taken in their absorption, we
must consider what tal<es place in the case of a few compound carbo-
hydrates which form an important part of our food. We refer to milk-
sugar which occurs in milk, and the numerous carbohydrates other than
starch that are olitained in vegetable nutriment, cellulose especially.
Milk-sugar is unquestionably decomposed into its' components while in
the bowels in the case of animals accustomed to milk nourishment, espe-
cially during the age of suckling.' On the other hand, in many animals
there seems to be no ferment present in the whole of the alimentary canal
which is capable of splitting up milk-sugar. What happens to milk-sugar
in such cases is not clear to us at present; probably it is further decom-
posed in the wall of the canal. The question as to the utilization of car-
bohydrate introduced into the organism in the form of cellulose is a very
important one. Cellulose plays no part at all in the nourishment of the
carnivora, and it is also unessential in the case of the omnivora, whereas
in the case of the herbivora a not inconsiderable part of the carbohydrates
contained in their nourishment is in the form of cellulose. Now, this
compound is not acted upon by the saliva, nor by the juices of the stomach,
pancreas, or intestine, provided we leave out of consideration the action
of bacteria which are ever-present. It is here that the activity of certain
micro-organisms present in the intestines comes into play, and this forms
to some extent an example of symbiosis. The fact that cellulose is actually
subject to a transformation in the bowels is proved by our being unable
to find in the faeces the whole amount of cellulose which has been introduced
into the system.^ Outside of the organism, it has been found possible to
dissolve as much as seventy per cent of cellulose by means of the intestinal
juices from a horse; these are rich in bacteria.' Sugar is not formed by
this process, but a considerable amount of gas is evolved. The mixture
produced by the decomposition has an acid reaction. These products
have been studied by Tappeiner.^ He found that by the action of meat

' Cf. Rohmann and Nagano: Pfliiger's Arch. 95 60 (1903); Ernst Weinland: Z.
Biol. 38, 16, and 606 (1899); 40 (1900).

' The food-vahie of cellulose has not been determined definitely even as regards the
herbivora. Cf. the following articles: — W. Henneberg and F. Stohmann: " Beitrage zu
einer rationellen Futterung der Wiederkauer." Braunschweig, 1S60 and 1864. Z. Biol.

21, 613 (1885). V. Knieriem: ibid. 21, 67 (18S.5). Weiske, Schulze, and Flechsig: ibid.

22, 373 (1886). E. Wolff: Landwirtsch. Jahrbiicher, 49, Suppl. Ill (1887). N. Zuntz:
Pfliiger's Arch. 49, 477 (1891).

^ Viktor Hofmeister: Arch, wissensch. und prakt. Heilkunde, 11, 1 and 2 (18S5).
* Z. Biol. 20, 52 (1884); 24, 105 (1888); and Hoppe-Seyler: Z. physiol. Chem. 10, 401
(1886).

CARBOHYDRATES. 63

extract which he had inoculated with bacteria from the contents of the
paunch upon absorbent cotton-wool, the following products were formed:
carbonic acid, methane, and fatty acids (acetic, butyric, and valeric
acids). It is perfectly possible that the breaking down of the cellulose
takes place similarly in the intestinal canal. The behavior of cellulose
here has, however, never been entirely explained. It is also possible that
perhaps only a portion of the cellulose is decomposed in this way, while
another portion may be acted upon differently perhaps by means of the
epithelium of the canal itself, being transformed in such a way that it can
be absorbed. Not only the herbivora are capable of utilizing cellulose, but
the omnivora can make use of it at least to some extent. The investiga-
tions of V. Knieriem have shown that the human intestine is capable of
dissolving a part of the tender cellulose from young vegetables. As much
as forty per cent of the cellulose introduced into the system could not
be detected in the faeces.'

Cellulose, especially in animals possessing a long intestine, — chiefly
the herbivora, but also the omnivora, - — plays still another characteristic
part, as the following experiments show. If rabbits are fed with food
containing no cellulose, they soon die. This is due to the fact that
when cellulose is left out of the nourishment the intestine no longer expe-
riences a certain mechanical irritation to which it has become accustomed.
On this account the peristalsis becomes retarded, then the contents of the ^^
intestines accumulate, whereby putrefaction ensues, and eventually there ^

is inflammation of the bowels. That this explanation is correct, is shown
by the fact that the animals experimented with continue to live, if, instead
of the cellulose, the animals are fed with horn shavings which are perfectly
indigestible.-

The bacteria contained in the stomach and intestines attack not only
cellulose but other carbohydrates as well. For this reason, the breaking
down of the more complicated carbohydrates does not actuall}' take place
so simph' as has been depicted. On the other hand, the decomposition
brought about b}' means of bacteria is, in general, not very extensive, and
depends very much upon the external conditions. The products formed by
their action are chiefly lactic acid, formic acid, acetic acid, butyric acid, and
alcohol with evolution of carbon dioxide, hydrogen, and methane.' There
are, furthermore, other micro-organisms known, as, for example, Bacterium *
thermn, which break down starch in very much the same way as this is
accomplished by the diastase in saliva and pancreas, thus aiding the
conversion of amvlum into sugar.

V

Tmc. cU.

von Knieriem: loc. cit.

Cf. H. Tappeiner: Z. Biol. 19, 228 (1883).

J. Wortmann: Z. physiol. Chem. 6, 287 (1882).

64 LECTURE IV.

The question has often been raised as to whether the micro-organisms
of the aUmentary canal are absolutely necessary for the perfect digestion
of the food, or whether they should be regarded as true parasites. To
decide this point, Nuttall and Thierfelder ' have made the following experi-
ment: By Csesarean section, they removed guinea pigs from the uterus
of the mother shortly before their normal birth, taking most careful anti-
septic precautions, and placing them in a sterilized cage. Guinea pigs,
unlike most of the related animals, come into the world in such a developed
state that they are able to assimilate properly the I'ood of the adult. It
was found possible, as proved by later examination, to keep these little
pigs perfectly sterile during the entire experiment (eight days) and to feed
them with sterile food, crackers and milk, in a sterile environment. The
animals experimented with gained in weight normally, thus proving that
the animal organism could thrive when bacteria were absent. These
experiments were especially valuable because they were performed with
animals which are particularly likely to be infested with bacteria from
their vegetable food. It may be said, on the other hand, however, that
the experiment merely shows that guinea pigs can subsist upon crackers
and milk in the absence of bacteria, but it does not necessarily follow that
the result would have been the same if a food rich in cellulose had been
fed them.

Schottelius ' has arrived at quite different results from those of Nuttall
and Thierfelder. He chose for his experiments chickens which were
hatched under sterile conditions, kept in sterile places, and fed with sterile
food. These animals, although they ate abundantly, had continuous
hunger, and declined about as quickly as a starving animal. As soon as
bacteria from hen fteces were mixed with the food the animals revived and
increased in weight. Recently Moro ' has carried out similar experiments
with the larvae of the mud-frog {Pelobates fuscus). He was able to keep
them sterile for thirty-six days. It was found, however, that if the sterile
larvae were placed in water containing the fsces of the mother, the
increase in weight and general development was much more rapid than
was the case with larvae kept sterile.

In discussing the carbohydrates we have mentioned the fact that the
five-carbon sugars, the pentoses or their condensation products the pen-
tosans which are so widely distributed in the vegetable kingdom, are not
unimportant forms of nutriment, especially in the case of the herbivora.
The researches of Stone ^ and of Weiske ^ have shown that the herbivora

■ Z. physio!. Chem. 21, 109 (1895-96); 22, 62 (1896-97).

' Arch. Hyg. 34, 210 (1899). and 42, 48 (1902).

■'' Jahrb. fiir Kinderheilkunde, 62, H. 4 (1905).

* Am. Chem. J. 14, 9 (1902).

' Z. physiol. Chem. 20, 489 (1895).

CARBOHYDRATES. 65

can utilize up to fifty or sixty per cent of the pentosans contained in
vegetables. In these experiments a mixture of pentosans (araban,
xylan, methylpentosan) was fed to the animals, but recently Slowtzow '
has fed pure xylan to rabbits. In the excreta from 17.1 to 66.8 per cent
of the pentosans were found in an unchanged condition. The remainder
was undoubtedly utilized in the system after having been converted into
the simpler sugars (pentoses).

From the intestine on, the simple sugars (e.g., d-glucose) are quickly
absorbed, whether introduced into the system in this form, or obtained
from the destruction of more complicated sugars. There are two ways in
which the nutriment absorbed by the intestine can reach the general cir-
culation. In the first place it can enter directly by means of the blood-ves-
sels, in this case the branches of the portal vein. This is the path taken by
salts, carbohydrates, and proteins. In this way they reach the liver, there
to undergo certain important transformations, aftJer which they are capable
of being introduced into the general circulation of the blood. The second
path is by way of the lymphatics, which conduct the alisorbed sub-
stances, especially the fats, into the thoracic duct, from which they are led
into the ]'ena anonyma, and thus into the general circulation.

The experiments showing that the absorption of carbohydrates as a
matter of fact takes place in the first manner will be discussed later when
we come to consider the absorption of fats.

In order to get some idea of the process by means of which sugar is
transferred to the circulatory system, and in order to understand how
large amounts of carbohydrates, e.g. 500 grams, can be transferred in a
relatively short time to the blood-stream (especially into the portal vein)
without materially increasing the sugar content of the blood, we must
remember what an enormous surface for absorption is presented by the
extremely fine network of blood-capillaries. Absorption takes place
continuously hand in jaand with the breaking down of the more com-
plicated carbohydrates into simpler sugars. • Although the tiny molecules
of sugar are absorbed at thousands of places and pass into the blood, and
although they are immediately carried awaj', it would seem that there
must be an increase in the amount of sugar contained in the blood corre-
sponding to the amount absorbed. That this is not the case — the normal
amount of sugar is 0.5 to 1.5 grams per liter and remains constant — must
be due to the fact that sugar is removed from the blood to the same extent
that it is absorbed by it. This is, as a matter of fact, exactly what hap-
pens, and it is the liver which regulates the exchange of carboh}'drates in
the whole animal system. It intercepts the absorbed sugar, and keeps the
sugar content in the lilooil constant. By means of the activity of the

' Z. physiol. Clipm. 34, 181 (1901). See also Rudziiiski : ibid. 40, 317 (1904).

66 • LECTURE IV.

liver cells, the molecules of d-glucose are made to unite together again
with loss of water and the formation of a new polysaccharide, glycogen.
In making this transformation the animal organism acts precisely like the
plant in forming cellulose, the circulating sugar being removed from
metabolism, stored up and protected from combustion in such a way
that at any given moment it can be made " liquid " again. This stage
in the metabolism of carbohydrates in the animal system can be demon-
strated very simply by means of the following experiment: A number
of rabbits may be kept from food for a length of time until it is known
as a matter of experience that the glycogen content of the organs, espe-
cially the liver, has been brought as low as possible. This is the case at
the end of about ten days. As we shall see later on, the consumption of
glycogen can be accelerated greatly by muscular work (whether by actual
body work — e.g. dogs running in a tread-mill — or by muscular convul-
sions produced by strychnine poisoning), and thus the time required for
the experiment greatly shortened. A part of the animals experimented
upon are then fed with a diet rich in carbohydrates. If now all the
animals are killed, those that are starving as well as those which have
been recently fed and are in the act of digesting the food, the former,
when subjected to quantitative tests, will be found to contain only
traces of glycogen in the liver, whereas the latter will contain a large
amount.' This state of affairs is of regular occurrence, so that to-day
there is scarcely any one who doubts that there is a direct connection
between the carbohydrates taken up (the absorbed d-glucose) and the
glycogen.^ The fact that the liver actually forms glycogen directly from
sugar has been demonstrated recently by Karl Grube.' Grube passed blood
rich in sugar through the liver of a dog, and could detect a slight increase
in the liver-glycogen. It is another question as to whether all carbo-
hydrates, for example, the pentoses, are capable of forming glycogen in
this way. We shall later on take up this point more in detail, but at this
place it is of interest for us to know merely how the more important carbo-
hydrates in food, namely, starch, cane-sugar, and grape-sugar, behave in
this respect. Starch, as we have already seen, is decomposed eventually
into molecules of d-glucose, and the same is true of saccharose. From the
latter, however, not only rf-glucose but an equal amount of d-fructose is
formed, the latter being a ketohexose. The question that now arises is,

' Cf. F. W. Pavy: Phil. Transact, for 1860, p. 579, and Researches on the Nature and
Treatment of Diabetes, London, 1862. Also The Physiology of Carbohydrates, 1894.
See also Pfliiger's Das Glycogen und seine Beziehung ziir ZuckerUrankheit, and Cramer's
Physiologie das Glykogens, Ergeb. Physiol. (Asher u. Spiro) 1, p. 803 (1902.)

' Carl Voit has furthermore shown that subcutaneous introduction of rf-glucose into
rabbits caused an increase of up to eight per cent glycogen in the liver. Z. Biol. 28,
245, 288 (1891). See also Erwin Voit: Z. Biol. 25, 551

^ Pfluger's Arch. 107, 490 (1905).

CARBOHYDRATES. 67

Does the latter become changed into glycogen, does it form a particular
glycogen of its own, or is the fructose first changed into d-glucose,' and
then, in common with the other glucoses, changed to glycogen? Until
recently it was quite generally assumed that the last-mentioned process
was carried out, but now certain facts have become known which will
perhaps lead us to another conception. It has been established that,
after the e.xtirpation of the pancreas in dogs, not only does sugar appear
in the urine, but at the same time the formation of glycogen in the liver is
interfered with, and as a matter of fact this disturl)ance is much more
pronounced when rf-glueose is fed to the dog than in the case of fructose.
The exact significance of this result is at present not clear.

When the food is rich in carbohydrates the liver cannot retain all of
it as glycogen. The glycogen stored in the liver of man amounts at the
most to 150 grams. The muscles can take up an equal amount provided
they did not originally contain considerable. As this is often the case
under normal conditions, however, we must answer the question as to
what becomes of the sugar which is not disposed of as glycogen. A direct
consumption of large amounts of sugar is not to be thought of; and on the
other hand the sugar content of the organs, and of the blood especially,
never exceeds certain well-estalilished limits when the storage places for
glycogen have been filled. Here for the first time, we meet with the
question of the transformation of one food-stuff into another. Sub-
sequently we shall have to study this closely, but at present we will merely
mention that the excess of sugar is evidently disposed of as fat, a phenom-
enon which we meet with in the vegetable kingdom, and one which plays
an important part in the depositing of nutriment in latent seeds, and
conversely in its utilization at the time of germination.

Now what becomes of this stored-up glycogen? As we have seen, the
glycogen gradually disappears if nourishment is withheld or work is
performed. It was Claude Bernard ^ who first showed this relation between
glycogen and muscular work. He found that the livers of hibernating ani-
mals during their winter's sleep contained large amounts of glycogen, and
not only was the glycogen contained in the liver cells, but also in the
muscular tissue and in the lungs. As soon as the animals awoke and

' Such a transformation is explained to u.s by the work of C. A. Lobrj' de Bruyn and
W. Alberda van Ehenstein, Her. 28, .'5078 (18!).')), and Kec. trav. cliim. 14, 103, 15G.
The.-ie two authors have shown that phico.se, fructose, and raaimose can be easily trans-
formed into one another in alkaline solution. The transformation of mannose into
glucose is equally interesting as that of fructose into glucose, for the former is used
as material for the formation of glycogen. Thus in Japan a natural manna is found
which serves the inhabitant in the same way as starch does for us. f'f. Low and Tsuji:
Landwirtschaftliche Versuehstationen, 45, 433. Also Haycraft: Z. physiol. Chem. 19,
137 (1894).

' Compt. rend. 48, 673 (1859).

68 LECTURE IV.

began to move about, Bernard noticed that the glycogen disappeared.
He furthermore observed that when the muscular tissues of well-nourished
mammals or birds were at rest — whether voluntarily so or as a result of
artificially severing the nerves in them — the glycogen content gradually
increased, only to disappear again when the muscles were set at work.

Direct experiments were carried out by S. Weiss.' He compared the
glycogen content of a frog's hind legs of which one had been tetanized
almost to exhaustion while the other was under control and rested. The
glycogen in the active muscles decreased from 24.27 to 50.43 per cent.
Finally Th. Chandelon ^ has carried out the following experiment: In a
rabbit he .severed the sciatic and crural nerves, and at the end of from 2
to 5 days found in the paralyzed muscles an increase in glycogen amount-
ing to from 5.51 to 172.4 per cent. Similarly Marcuse ' made similar
observations, and found the following glycogen values:

EXPERIMENT.

Per cent glycogen in the —

Unirritated Muscles.

Irritated Muscles.

I

Ill

0.748
0.749
0.589
0.542

0.657

0.539
0.461

IV

0 395

V

Average

0.341
0.434

Finally, the same result has been obtained by Edward Kiilz * in another
way. He caused a well-nourished dog to draw a heavy cart. The animal
weighed 45.500 kilograms, and was made to drag the cart for 9 hours
and 40 minutes. The dog was then bled to death. The glycogen deter-
mination showed the presence of 52.05.3 grams, i.e., 1.16 grams per kilogram
of the dog's weight. A well-nourished dog that is not tired shows a
glycogen content of 38 grams per kilogram. For comparison, it may be
mentioned that after 28 days of starvation a dog of about the same size as
the above-mentioned showed but 1.5 grams of glycogen per kilogram. It
is evident, therefore, that after about OJ hours of labor the glycogen stores
were consumed to fully as great an extent as in the case of a dog starved
for 28 days. Kiilz then repeated the experiment with three other dogs and
with the same result.

A further confirmation of the fact that the carliohydrates serve as an
important source of muscular energy is shown by the interesting experi-

Sitzber. Akad. Wiss. Wien. 64, Abt. 1.

Pfluger'3 Arch. 13, 626 (1876).

Pfiiiger's Arch. 39, 425 fl8S6). See also Edward Manche, Z. Biol. 25, 163 (1889).

Beitrage zur Kenntniss des Glykogens, p. 41 (1891).

CARBOHYDRATES. 69

ment of Fick and Wislicenus.' These scientists attempted to find out
what substances were chiefly decomposed as a result of strenuous mus-
cular work. At that time Liebig's - theory prevailed that the muscles
performed work at the cost of albuminous substances, and they decided
to test this experimentally. If Liel)ig's theory were correct, it was to be
expected that the elimination of nitrogen would be increased as a result
of muscular activity. Fick and Wislicenus climbed Mount Faulhorn, 1956
meters above the Lake of Brienz, which was the starting-point. For sev-
enteen hours before the start, during the ascent (which required six hours),
and for six hours following, care was taken to eat only food which was free
from nitrogen. All the urine passed during the ascent and the six hours
following was carefully collected and the nitrogen accurately determined.
From the results obtained by chemical analysis it was found that Fick had
decomposed 38.15 grams of albumin, and Wislicenus 37 grams. These
amounts of albumin correspond to about 250 heat units in each case, or
106,000 kilograms of work. If we estimate the actual work performed in
climbing the mountain we arrive at the following values. Wislicenus
weighed 76 kilograms. By simply raising this weight to the height of the
mountain peak, 76 X 1956 = 148,656 kilograms of work was performed.
These values suffice to show that the work could not have been at the ex-
pense of all)umin alone. This fact is still more striking when we remember
that the above value of albumin in heat units is too high, for it is based
upon the assumption that the carbon is completely changed to CO2 and the
hydrogen to H2O. Now, as a matter of fact, nowhere near this amount of
energy is obtained by the consumption of albumin in the animal organism,
for a part of the carbon, some of the hydrogen, and the greater part of the
nitrogen, are eliminated in the form of urea. In man, the amount of urea
formed is as a rule equal to one-third the weight of albumin decomposed.
Therefore from the above heat value of albumin we must deduct one-third
the heat of combustion of the same weight of urea. On the other hand,
as a matter of fact, the scientists performed much more work than we,
have estimated. Fick and Wislicenus estimate the amount of work per-
formed by their circulatory and respiratory apparatus as 30,000 kilogram-
meters. Furthermore, it must be taken into consideration that in every
motion, in every step, work is performed which is transformed into heat
and lost as far as the work performed is concerned. According to Helm-
holtz, only one-fifth of the actual heat of combustion is transformed into
external work. It is perfectly certain, therefore, that we can safely con-
clude from the above experiment that albumin alone is not the source of
muscular work, but, on the other hand, we are not justified in concluding

' Vierteljahresschrift dps Ziiricher naturforschenden Gesellsch. 10, 317 (1865).
' Chemisolie Briefe (1857). Ci. also Ann. 163 1, and 157 (1870); C. Voit: Z. Biol. 6,
305(1870); Schenck: Arch, e^^per. Path. Pharm. 2, 21 (1874).

70 LECTURE IV.

that the source is to be sought entirely in substances free from nitrogen.
At all events, however, Liebig's theory is untenable.

It remained for C. Voit ' to establish a more exact proof of the theory
that muscular work is performed chiefly at the expense of substances free
from nitrogen. He caused a dog to run in a tread-mill, and compared_the
amount of nitrogen in the urine which was passed during the working
period with that passed during rest, both before and after working. It
was found that the amount of nitrogen eliminated during twenty-four
hours of a working period was but slightly, if at all, in excess of that elimi-
nated during the resting periods. O. Kellner ^ tried a similar experiment
with horses, and obtained the same results when the animals experi-
mented upon were fed with an abundance of carbohydrates. If this was
not the case, the amount of nitrogen eliminated was considerably more.
Finally, Voit ' performed corresponding experiments with human beings,
and determined not only the amount of nitrogen eliminated, but at the
same time estimated the carbon dioxide, and indirectly the absorption of
oxygen.*

The increased absorption of oxygen and elimination of carbon dioxide
have also been observed in direct experiments upon the muscles themselves,
by comparing the amount of these gases contained in the venous blood of
a resting muscle and of one that has been tetanized.^ The inactive muscle
takes up more oxygen from the blood than it gives back to the latter in
the form of carbonic acid gas. Obviously the muscular cells retain oxygen
in some form or other. We can, in fact, speak of a storing up of oxygen.
This stored-up oxygen again appears after violent exercise, for then the
muscle gives up to the blood more oxygen as carbon dioxide than it takes up
from the blood as free oxygen. Yet it is also true that the muscle takes
up more oxygen from the blood during exercise than in periods of rest, for
venous blood contains during work less oxygen and more carbonic acid
than when the muscle is at rest. That evidently not only an oxidation
process but a hydrolytic decomposition as well should be regarded as the
source of muscular work will be shown later.

We must now answer the question as to how the glycogen is decom-
posed and in what way it is utilized in muscular work. Furthermore,
we are interested to know what connection there is between the principal

' Z. Biol. 2, 307 and 339 (1866).

' Landw. Jahrb. 8, 701 (1879), and 9, 651 (1880).

^ Z. Biol. 2, .307, 488 (1866).

* This was known to Lavoisier: Segiiin and Lavoisier: Mto. acad. Sciences, 688
and 696 (1789). See also Sond^n and Tigerstedt: Skand. Arch. Physiol. 6, 181 (1895).
O. Krummacher: Z. Biol. 33,117(1896) Zuntz, Frentzel, and Loeb: Arch (Anat.
und) Physiol. 541 (1894). Speck: *)(/. 465 (1895).

' Ludwig and Sczelkow: Sitzber. Akad. Wiss. Wien. 45, 171 (1862). Max v. Frey :
Arch. Anat. Physiol. 533 (1885).

CARBOHYDRATES. 71

store of glycogen, namely liver-glycogen, and the consumption of carbo-
hydrates by the muscles. According to all our experience up to the
present time, glycogen is not directly oxidized, but is previously decom-
posed into rf-glucose. This is shown directly by the experiments of J.
Ranke ' and of Otto Nasse." The former examined the sugar content of
the hind legs of a number of frogs, one leg being tetanized and the
other at rest. Several determinations showed 0.058 per cent sugar in
the dry substance of the resting muscle, and 0.082 per cent in the tetanized
muscle. The sugar content, therefore, was increased 41 per cent in the
latter case. The hydrolysis of glycogen is evidently caused, as Magendie '
has shown, by a ferment which in its action coincides with that of diastase,
which is already known to us. In the liver also the stores of glycogen are
decomposed in this way.* Here, besides (/-glucose, the intermediate products
dextrin and maltose have been detected. They are probably formed in
the breaking down of gh'cogen in the muscles, but their presence has never
been established with certainty. The fact that the hydrolysis of glycogen
in the liver is not to be considered as a result of the activity of the cells,
but rather that it is due to a ferment which can be separated from the
liver cells, was clearly shown as long ago as 1873 by von Wittich.^ He
proved that it was possible to extract by means of glycerol from liver
which was completely freed from blood and hardened by alcohol a fer-
ment which was capable of hydrolyzing glycogen, von Wittich showed
that this ferment belonged to the liver cells rather than to the blood by
again and again obtaining the diastatic ferment after repeated, thorough
washings of the liver. It is also possible, as Pavy has shown, to treat
liver with alcohol, dry it, and preserve it indefinitely. If such a preparation
is digested with water, the diastatic reaction will be obtained invariably.
On the other hand, the objection may be raised to this line of rea-soning,
that the formation of sugar is perhaps due to the action of micro-organisms,
an assumption which in the light of recent experience obtained by working
with organs and their extracts does not seem improbable." E. Salkowski,'

' Tetanus, p. 168, Leipsic, 1865.

' Pfluger's Arch. 2, 97 (1869).

' Compt. rend. 23, 189 (1846).

' Miisculus and v. Mering: Z. physiol. Chem. 2, 416 (1878-79). E. W. Pavy: The
rhysiology of Carbohydrates, London, 1894. Kulz and Vogel: Z. Biol. 31, 108 (1895).

' Pfliiger's .Arch. 7, 28 (1873).

• For an explanation of the other view, that the sugar formation from glycogen is
caused by the life proce.ss of the liver cells, see M. Foster: Text-book of Physiology,
appendix by Sheridan I.ea, pp. 58, 98. Noel Paton: Hepatic Olycogenesis, Trans. Roy.
Soc. 1S94, and Phil. Trans. 185 B. 233 (1894).

' Deut. Med. Wochschr. No. 16, 1888; .\rch. Physiol. 554 (1S90); Zent. med. Wis-
sensch. Jg. 27, No. 13, 227 (1889). See also Otto Na.s.se: Rostockor Ztg, Nn. 105 (1889).
Salkowski: Z. klin. Med. p. 90 (ISOn, and Pfliiger's Arch. 66, 339 and 351 (1894).
Arthus and lluber: Arch, de Physiol. 651 (1892).

72 LECTURE IV.

however, has shown that this sugar formation will also take place in
chloroform water. Since aqueous solutions of chloroform prevent proto-
plasmic action including the action of micro-organisms, this experi-
ment proves that the formation of sugar from glycogen is not due to the
action of bacteria, and further that the liver cells themselves are not the
cause, but that the hydrolysis of glycogen is to be traced to the action of
a soluble ferment. Salkowski clearly established this important fact by
means of the following experiment: He removed the liver from a rabbit
which had taken into its stomach seventeen hours before death ten grams
of cane-sugar dissolved in water. After taking out the gall bladder and
the large bile ducts, the liver was cut up into fine pieces and triturated.
Two portions of the same weight of liver-pulp were taken, one of which
was placed directly in a bottle filled with chloroform water; the other
portion was boiled in water first, and then treated with the same amount
of chloroform water. After sixty-eight hours' digestion, the glycogen and
sugar were determined in each of the extracts. The first extract showed
a large amount of sugar and no glycogen, whereas the extract of the
boiled liver contained considerable glycogen and only very little sugar.
In the first experiment there were found 48.28 grams of sugar; in the second,
3.65 grams.

As we have seen, the glycogen content of the liver stands in a definite
relation to that of the muscles. After the muscles have performed hard
work, we find that not only does the glycogen in them disappear, but that
in the liver as well. This leads us to believe that the liver serves as a
central storage place which is capable of feeding all the other stores in the
organism. The transfer of the liver-glycogen takes place by means of
the blood, and in the form of rf-glucose as we have seen. Now the organism
strives to a remarkable degree, even during starvation, to maintain a
constant amount of sugar in the blood. If the glycogen in the muscles is
used up, the muscle cells then strive to form glycogen from the sugar in
the blood. This would cause a diminution in the sugar content of the
blood, except for the fact that as soon as this sugar is removed, then hepatic
glycogen is decomposed into d-glucose and removed by the blood.» It
has been stated that the higher products of the hydrolysis of glycogen,
namely dextrin and maltose, are likewise transported by the blood; but
how far such observations are correct, or what the extent to which this takes
place, cannot be decided at present. At all events, such statements have

' The assumption made liy J. Seegen CDie ZuckerbildunRim Tierknrper, ihr ITmfang
und ihre Bedeutung;, Berlin, LSQO, and Studien iiber Stoffwechsel im Tierkorper. Berlin,
1887), that blood-sugar is formed from proteins in the nourishment, and that on the
other hand the liver-glycogen originates from the fats, is not supported by the facts.
Cf. R. Bohm and F. A. Hoffmann: Pfliiger's Arch. 23, 205 (1880). H. Girard: Pfluger's
Archiv. 41, 294 fl.S87). E. Cavazzani: Arch. Anat. Physiol. 539 (1898).

CARBOHYDRATES. 73

lost much of their significance since it has become l^nown that the blood-
serum contains a ferment which is capable of converting glycogen and
starch into dextrose.' The fact that the muscles are capable of forming
glycogen from glucose has been proved by Kiilz.- By subcutaneous
injection of sugar into frogs with extirpated livers he was al)le to establish
the fact that there was an increase of muscle-glycogcn.

That the sugar content of the blood is directly dependent upon the liver
is shown by the fact that ablation of the liver causes the amount of sugar
in the blood to diminish and finally disappear.^

Although we have outlined the way carbohydrates break down in the
alimentary canal, the absorption of their hydrolytic products and their
destiny in the animal organism from the time of their being stored up as
glycogen on to their change back into d-glucose, still we have failed to give
an exact picture of the manner in which the glucose formed is eventually
consumed. We are, indeed, acquainted with the end-products, carbon
dioxide and water, and know that an oxidation takes place, but we are still
in doubt concerning the intermediate products. The destruction of the
sugar has been traced by Lepine ^ and others to the action of a glucolytic
ferment in the blood and in the tissues. Claude Bernard ^ had previously
shown that the sugar content of blood gradually diminishes on standing.
More recently it has been found that ferments of similar action are present
in almost all of the organs. It is hard to decide whether in all cases there
is no cooperation of micro-organisms, and as to what part this decom-
position of d-glucose plaj's in the living tissue. At all events, there is at
present no justification for the assumption that all of the sugar decomposi-
tion is caused by the action of the ferment mentioned. In this connection
we will refer to the work of Stoklasa." He obtained from the expressed
juices of all sorts of different organs (muscles, liver, lungs, pancreas) by
precipitation with alcohol-ether, ferments which produced alcoholic fermen-
tation in a sterilized sugar solution without the aid of bacteria. The
proportion of carbon dioxide and alcohol formed was the same as in the
fermentation brought about l\y the zymase in yeast. The decomposition

• M. Bial: Pfliiger's .Vrcli. 52, 1.37 (1.S92), and 54,73 (1<S9.3). Cf. also Rohmann,
Ber. 25, 36.M (1802).

' Pfliiger's Arch. 24, 61 (1881).

' Cf. Tang! and Harley: Pfliiger's Arch. 61, 551 (1895). Pavy and Siau: J. Physiol.
29, 375 (1903). Minkowski: Arch. exp. Path. Pharm. 21, 41 (18S6). Schenk: Pfliiger's
Arch. 57, 553 (1S94V

« Compt. rend. 110, 712 flS90): 110, 1311; 112, 146 (1891); 112, 411 ; 112, 604; 112,
1185 and 1414; 113, 118 (1891); 120, 1.39 (1895V L«?pine: Le ferment plycolytique et la
pathog^niedudiabote. Paris, 1891. Cf . Nasse and Framm : Pfliiger's Arch. 63, 203 (1896).

^ Le(;ons sur le diabete (1878).

« Hofmeister's Beitr. 3, 460 (1903); Ber. 36, 4058 (1903) ; Pfliiger's Archiv. 101, 311
(1904); Zentr. Physiol. 17, 465 (1903); and Ber. 38, 664 (1905).

74 LECTURE IV.

of the carbohydrate did not stop with the formation of the products
named, but acetic and formic acids were formed with consumption of
oxygen. It is at present hard to tell what part the alcoholic fermentation
plays in the living organism, or whether, in fact, it has any significance.
At all events, Stoklasa's observations include the possibility of energy
being produced without oxygen being supplied. The fact that the animal
organism evidently makes use of such simple decompositions as a source of
energy is shown by the interesting experiments of Hermann, Pfliiger, and
Bunge. Hermann ' proved that a piece of extirpated muscle, from which
no more oxygen could be pumped out, could work in an atmosphere free
from oxygen and produce carbonic acid. Hermann at the same time
detected the formation of an acid (lactic acid). Pfliiger^ succeeded in
keeping a frog alive for twenty-five hours at a temperature a few degrees
above the freezing-point of water in an atmosphere free from oxygen,
during which time the animal evolved a considerable amount of carbonic
acid gas. Finally G. V. Bunge' showed that a parasitic-worm of the cat,
Ascaris mystax, could survive for four or five days in a medium absolutely
devoid of oxygen, and move around in a very lively manner during that
time. It should by no means be concluded from these very interesting
experiments, that the animal organism performs its muscular work solely
at the expense of the energy set free by hydrolytic decompositions.
The amount of living force produced in this way would be altogether too
small. On the other hand, it is conceivable that the cell, by means of a
partial breaking down, that is to say, by a hydrolysis and subsequent
oxidation of the decomposition products, can increase when necessary the
amount of energy available. Thus 100 grams of glucose when completely
oxidized to carbon dioxide and water yield 39.39 calories (= 1,674,000
kilogram-meters of work). By alcoholic fermentation, i.e., the hydrolysis
of 100 grams glucose into carbon dioxide and ethyl alcohol, only 372
calories(= 158,100 kilogram-meters of work) are liberated.

As a result of work the muscular tissue, which is amphoteric, becomes
of acid reaction. This change is, at least to some extent, due to the
formation of lactic acid. Formerly it was believed that there was a direct
connection between the formation of the latter and the decomposition
of carbohydrates. More recently, however, this view has been strongly
coml)ated. At present it has not been definitely decided as to what
relation the lactic acid bears to the performance of work by the muscles.
It is possible that its presence is due to a hydrolysis caused by the
absence of a sufficient amount of oxygen. On the other hand, it is also
conceivable that the formation of lactic acid has nothing whatever to do

' Untersuchungen iiber den Stoffwechsel der Muskeln, Berlin, 1867.

' Pfliiger's Arch. 10, 251 (1875).

' Z. physiol. Chem. 8, 48 (1883-84) ; 12, 565 (1888); 14, 318 (1889).

CARBOHYDRATES. 75

with the combustion of carbohydrates in the muscle, but results from the
breaking down of protein. We shall see later on that alanine, which
is formed by the hydrolysis of albumin, stands in close relation to lactic
acid.'

The role of the carbohydrates in the animal organism is by no means
limited to the production of muscular energy. Above all they are to be
considered as a source of heat. Thus it is possible to cause the glycogen
stores to disappear by merely chilling the animal.- The carbohydrates,
without doubt, take an active part in the life process of the individual
cells. They take part also in their building up. At present we know
nothing concerning the way they occur in the organism and concerning
their union in the cell complex. We have already mentioned the occur-
rence of pentoses, especially xylose, which is in the nucleoproteids. Wc
shall later on see that there are indications of the hexoses also being used
as building material for the nuclei of the cells.

' Cf. Astaschewsky: Z. physiol. Chem. 4, 397 (1880). Warren: Pfliiger's Arch. 24,
391 (1881). Heffter: Arch, exper. Path. Pharm. 31, 225 (1893). Werther: Pfluger'a
Arch. 46, 63 (1890). Spiro: Z. physiol. Chem. 1, HI (1877-78). Zillesen: Z. physiol.
Chem. 15, 387 (1891). Araki: Z. physiol. Chem. 15, .33.5 and .546 (1.S91), and Z.
physiol. Chem. 16, 453 (1892). Hoppe-Seyler: Z. physiol. Chem. 19, 476 (1894).

' E. Kulz; Pfluger's Arch. 24, 14 (1881).

LECTURE V.

CARBOHYDRATES.

IV.

Building Up and Breaking Down of Carbohydrates in the
Animal Organism.

We found in the last lecture that the blood under varying physiological
conditions always maintained a constant sugar-content. This does not
increase when the food is rich in carbohydrates, nor decrease materially
during starvation. This phenomenon is explicaltle only by the assumption
that there is an extraordinarily delicate regulating mechanism which
works on the one hand in conjunction with the organs containing the
stored-up sugar, and on the other hand with the places where sugar is
consumed. If for any reason the amount of sugar in the blood increases
above the normal, then sugar appears in the urine. This may take place,
for example, when an excessive amount of sugar has been introduced into
the alimentary canal. In such a case it is not always possible to remove
the sugar fast enough from the general metabolism by converting it into
glycogen or fat. This ability of storing up sugar is a restricted one.'
The maximum amount of sugar which it is possible for the system to
assimilate is known as the limit of assimilation, and the elimination of
sugar which takes place when this is exceeded is spoken of as alimentary
glucosuria. The limit varies with different forms of sugar and for different
individuals. In general, the danger of giving the blood an over-supply of
sugar under normal conditions of nourishment is but slight, because under
normal conditions the bulk of the carbohydrates are taken into the sj^stem
either as starch or as cane-sugar. There is no danger of these forms of
sugar being suddenly broken down in the alimentary canal; on the con-
trary, their decomposition always takes place gradually from one stage to
another so that this in itself serves in a measure to regulate the absorption
of sugar.

Under certain conditions the assimilation limit for carbohydrates can
be reduced greatly for a time. This is particularly true of the central
organ of carbohydrate metabolism, namely the liver. Claude Bernard '
recognized the fact that the storing up of sugar in the form of glycogen

' Cf. Franz Hofmeister: Arch. exp. Path. Pharm. 25, 240 (1889).
* Lemons (Cours du semestre d'hiver), 1854-55, p. 289.

76

CARBOHYDRATES. 77

and its transformation into glucose, both changes being functions of the
liver cells. — partly directly and partly indirectly, — were dependent upon
the nervous system. He showed this by means of the following classical
experiment, which was first performed upon rabbits. If a rabbit is injured
at a certain place in the medulla, sugar appears in the urine after a
short time. This region is bounded above by the origin of the two audi-
tory nerves, and below by a line connecting the places of origin of the
vagus nerves. The experiment is carried out in this way: After tying
the rabbit, the point of a trocar is placed in the median line upon the
OS occipilale exactly at the Protuberantia occipitalis superior, and then
pushed through carefully until the Pars basillaris is reached. The instru-
ment thus bores through the skull, the cerebellum, and the posterior
and median columns of the medulla. Two hours after the operation
sugar appears in the urine. The elimination of sugar, however, in such
cases is not lasting, usually disappearing at the end of five or six hours.
With dogs, however, this glucosuria lasts longer. Claude Bernard in one
case found it to last for a week. In such cases the amount of sugar con-
tained in the urine is not large, usually amounting to merely two or three
per cent.' Bernard shows the cause of the sugar elimination to be an
excessive amount of blood-sugar. He found, instead of the customary
0.1 to 0.1.5 percent, more than 0.3 per cent. This operation also has the
same result when performed with birds - or with frogs. ^

An observation made by F. W. Dock * is of considerable importance for
complete understanding of this kind of glucosuria. He found that the
above operation succeeded only with well-nourished animals, i.e., with those
which po.ssessed stored-up glycogen. Naunyn ^ arrived at the same con-
clusion. He showed that the success of the so-called " diabetic puncture "
depended wholly upon the state of nourishment of the animal experimented
upon. Dissection of the animal after the operation always showed that the
liver was free from glycogen. The fact that the liver in this case loses the
power of storing up sugar in the form of glycogen is shown by the following
experiment: If a solution of rf-glucose is injected into the mesenteric
vein of a dog whose liver has been deprived as far as possible of glyco-
gen by starvation, only very small amounts of sugar will subsequently
be found in the urine. If the same experiment is repeated with another
animal which has undergone the " diabetic puncture," a marked glucosuria
ensues soon, after the injection of the sugar."

' n^don; Diabete, Uictionnaire Je Physiol. 4, 812.

' M. Bernhardt: Virohow's .Vroh. 59, 407 (1874).

' M. Schiff: I'ntcrsiichungen iiber die Zuckerbildung, Wurzburg, 1859.

' Pfluger's .\rchiv. 5, ,571 (1872).

' Arch, exper. Path. Phann. 3, 85 (1875).

• Cf. P. Levine: Zentr. Physiol. 8, .397 (1894).

78 LECTURE V.

The question now arises as to what the connection is between the
" diabetic puncture " and the flooding of the organism with sugar. Claude
Bernard, by means of the following experiment, showed that the vagus
nerves participate in this.' If the " tUabetic puncture " is performed
after cutting these nerves at the neck, it is as effective as when the nerves
remained intact. On stimulating the peripheric stumps of a vagus nerve,
no glucosuria ensues. It appears immediately, however, if the central end,
i.e., the end connected with the medulla oblongata, is stimulated. In
such experiments Bernard showed that the whole body of the animal
experimented upon was flooded with sugar. The greatest quantity was
found in the hepatic veins. Bernard showed, furthermore, that cutting
the vagus nerves at the neck resulted in making the liver free from sugar.
He concludes from all these experiments that in the medulla oblongata
there is a center which regulates the transformation of sugar by the liver.
Communication, i.e., the excitement, is provided by means of the vagus
nerve. The sugar formation caused by stimulation of the central stump
of the vagi nerve, Bernard conceives to be a reflex action;^ and it is the
pulmonary branches of the vagus which contain the fibers acting upon
the sugar center, for if the vagi are cut above the liver and below the
lungs, no influence upon the sugar formation of the liver was observed.

Now how does this sugar center exert its influence upon the liver?
Bernard cut through the spinal cord at different places below the medulla
oblongata, and found that the path of communication must lie in the
upper parts of the spinal cord ; for if it was severed below the lower dorsal
vertebrse, the sugar formation in the liver was not affected. C. Eckhard,'
who confirmed this conclusion, found that after severing both the vagi
and sympathetic nerves in the neck, a successful diabetic puncture could
be made. After severing the splanchnic nerves, however, it ceased to be
operative. This indicates that the diabetic puncture acts uf)on the trans-
formation of the carbohydrates in the liver by nerve impluses passing
along the path of the splanchnic nerves.

We must assume, therefore, that the sugar formation in the liver is
regulated directly by a center in the medulla oblongata. The vagi nerves
conduct the centripetal impulses, and the splanchnic nerves the centri-
fugal. Later on, when we come to discuss digestion, and especially
the dependence of the secretion of the digestive glands upon certain
nervous influences, the above hypothesis will no longer appeal to us as
remarkable.

Thus far we have left the matter unsettled as to whether the liver alone
gives up sugar after the "diabetic puncture," or whether the sugar in the

> Cf. C. Eckhard: Beit. Anat. Physiol. 8, 77 (1879).
= Cf. E. F. Pfliiger: Das Glykogen, loc. cil. 386.
' Beitr. Anat. Physiol. 4, 138.

CARBOHYDRATES. 79

urine also comes from other organs. The experiments of Moos' and of Moritz
Schiff- have proved that only the sugar formation in the liver is affected.
If the vessels of the liver are all tied, the diabetic puncture becomes
inoperative. This is particularly well shown by the experiments of Schiff,
who took eight frogs of the same size, and produced glucosuria in all of
them. At the end of from two to four and three-quarters hours sugar
could be detected in the urine. The livers of all the animals experimented
upon were then exposed, drawn out through the abdominal wound, and
all the vessels and bile ducts encircled with a slip-knot of thread. In
four cases the knots were drawn tight, while in the other four they were
not. In the latter ca.se the glucosuria continued, while in the former it
gradually diminished, so that at the end of three hours the urine was free
from sugar.

It is not yet clear how this increased sugar formation is brought about.
There are several conceivable possibilities. The fact that the glycogen
stored up in the liver is suddenly converted into glucose, would make it
seem probable that the diastatic action has been consitlerably increased
above the normal. It is also conceivable that there is an increased pro-
duction of diastase, or, on the other hand, it is possible that under
normal conditions the glycogen is not, as ordinaly assumed, deposited in
the cells as a foreign body, but rather in the form of a loose chemical
combination, and that the diasta.se begins to act upon glycogen as soon
as it is set free. By exerting certain influences upon the liver cells, —
whether by the diabetic puncture, or whether by some other excitement
of the sugar center, — all of the glycogen is perhaps .set free from its
loose chemical combination, and subjected to the action of diastase, which
found no point of attack as long as the glycogen was in a combined
state. Claude Bernard believed that the increased formation of glucose
was due to an increased blood flow, which was proiiablj' caused by the
fact that the diabetic puncture had an effect upon the vaso-motor center.
He had in mind the increased blood flow which accompanies the increased
secretion of saHva by the submaxillary^ gland on stimulating the fibers
in the chorda tympani. R. Heidenhain, however, has shown that an
increa.sed secretion of saliva may take place without an increase in the
blood flow, and that the chorda tympani evidently possesses certain specific
secretorj- fibers. .Analogously, it is possible that the splanchnic nerves
contain fibers which exert an influence upon the formation of glucose in
the liver.

Closely connected with the so-called "diabetic puncture" we have
another observation to consider. If a one per cent, solution of common

' Arch, wisspnschaftl. Heilkunde, 4, 37.

' Untersuchungen iiber die Zuckerbildung in der Leber, p. 76, Wurzburg, 1859.

» Pfliiger's Arch. 6, 309 (1872); 9, 335 (1874).

80 LECTURE V.

salt ' is introduced into the vascular system, glucosuria ensues. If, on
the other hand, the splanchnic nerves are severed, the salt infusion
becomes ineffective. Morphia - behaves similarly. Quite recently the
exact connection between these experiments and the glucosuria produced
by the " diabetic puncture " has been developed by Martin H. Fischer." He
showed, first of all, that instead of the one-sixth-molar solution of common
salt which he injected into the^ circulatory system of rabbits at the rate of
75 to 100 c.c. per minute, one-sixth-molar solutions of other sodium salts
— e.g., NaBr, Nal, NaNOa — could be used with the same effect.'' Fischer
showed, furthermore, that the cause of the glucosuria was not a per-
manent injury produced in any organ, for he could cure the disease
by the use of a solution of calcium chloride. A renewed injection of the
common salt would, however, again lead to glucosuria. The greater
the concentration of the solution of sodium salt employed, the sooner
the sugar appeared in the urine.

Fischer, now, sought to determine upon which tissues the solution of
salt acted in producing this glucosuria. He remembered at the same time
the experiments of Kiilz, who observed that glucosuria was only produced
when the splanchnic nerves remained intact. Fischer found that after
cutting these nerves, he could not produce glucosuria, and, furthermore,
if the glucosuria was already established before the nerves were severed, it
would disappear shortly after the operation. This suggested the thought
that probably the salt solution produced some such action as that pro-
duced by the vagi nerves upon the sugar center. Fischer sought to localize
the action of the common salt as much as possible. For this purpose he
tied the axillary artery in some rabbits, and injected the solution of salt
into the central end of this artery so that the salt passed on through the
vertebral artery directly to the medulla ol^longata. When the salt solu-
tion was injected in this way, the sugar appeared in the urine somewhat
more quickly than when the same amount of the same solution was intro-
duced into a peripheral vessel. Furthermore, the glucosuria was more
severe and was moi-e lasting. As much as 7.3 per cent of urine sugar
was found in the urine. These experiments make it seem probable that
the salts introduced into the circulation act upon the same center as that
of the " diabetic puncture."

There are other poisons known which will produce glucosuria, strychnine,
for example. The poisonous effect of this substance is not obtained,
however, if the spinal cord, with the exception of the upper portions, is
extirpated. Since strychnine does not cause elimination of sugar in the

' C. Eckhard: Beit. Anat. Physiol. 8, 77 (1S79).

' Kiilz: Eckhard's Beitrage, 6, 177 (1872),

» University of California Publications, Physiology, 1, 77 (1903), and 1, 87 (1904).

*.LiCl, KCI, and SrCl^, produced glucosuria; NH^Cl did not.

CARBOHYDRATES. 81

urine of frogs with extirpated livers, it is probable that the glucosuria is
to be attributed to a disturbance of the liver function. It might be sup-
posed that the sugar elimination in the urine is a result of the tetanus
produced l\ythe strychnine poisoning. That this is not the case is shown
by the fact that animals poisoned with large amounts of strychnine — large
doses, instead of producing tetanus, cause paralysis of the motor nerves —
are also subject to glucosuria, and, as a matter of fact, in a more severe form
than in the case of animals with tetanus, caused by smaller doses of strych-
nine. The simplest explanation of this last fact is that during tetanus
sugar is consumed. Other poisons, such as phosphorus, arsenic, uranium
salts, corrosive sublimate, carbon monoxide (illuminating-gas), amyl
nitrite, curari, chloral, nitrobenzene, chloroform, acetone vapors, ether,
etc., will also cause glucosuria.' At present we know nothing more definite
concerning the mode of action of these various substances. It should not,
however, be assumed that they all have the same point of attack.

The glucosuria produced by the glucoside phloridzin has been stutlied
with especial thoroughne.ss.- If from one to three grams of this sub-
stance is administered to a dog for each kilogram of its body weight,
glucosuria results. In the casfe of starving animals, after the injection
of from O..*? to 2.5 grams of phloridzin, the sugar can be found in the
urine. Von Mering, the discoverer of this form of glucosuria, also studied
the effect of the decomposition products of phloridzin, and found that
phloretin alone was active in this respect, whereas phloroglucinol and
phloretic acid were inactive.

All the other glucosurias which we have discussed up to this point have
been caused by a glucohemia, i.e., a flooding of the organism and especially
of the blood with sugar. The elimination of sugar through the kidneys
is a result of a self-regulating mechanism. By eliminating the excess of
sugar the organism seeks to bring the sugar content of the blood back to
the normal. Even the glucosuria produced by phloridzin has been ex-
plained in this way,' although Mering himself and many other later inves-
tigators have failed to detect any increase in the amount of sugar contained
in the blood even when the ureter was tied. Phloridzin glucosuria is also

" Cf. Langendorff: Arch. Anat. Physiol. 1.38 (1887). F. Giirtler: Inaug. Dissert.
Kiinigsberg, 1886. Araki: Z. physiol. Chem. 17, .311 (1893). Luchsinger: Inaug.
Dis.scrt. Zurich, 187.5. Senff: Inaug. Dissert. Dorpat, 1869. Straub: Arch, exper. Path.
Pharin. 38, 1.39 (1897). Rosenstein: ibi,L 40, 363 (1898). Araki: Z. physiol. Chem.
16, .3.51 I1S91); 19, 4-J2, 476 (1894); also Bernard: Lemons sur la diabete et la gly-
cosurio animale. Paris, 1877.

' V. Moring: I'eber Dial)ete.s mellitus. Verhandl. Kongr. innere Med., 1886 u. 1887;
Z. klin. Med. 14, 405 (1888), and 16, 431 (1889). v. Mering and Minkowski: Zentr. klin.
Med. 10, 393 (1889). Max Cremcr: Z. Biol. 29, 175 (1893). Cremer and Ritter: ibid.
29, 2.56 (1893).

' Cf. Pa\-y: J. Physiol. 20, xix-xxii (1896). S. Leone: Gazz. internaz. di med. prat.
Vol. 3, 21.

82 LECTURE V.

shown to be different from the other forms by the investigations under
the leadership of Minkowski, carried out by Andreas Thiel ' and by von
Mering.- These investigators have shown that geese with extirpated
hvers ehminate sugar after the injection of phloridzin, whereas we have
seen that with the other forms of glucosuria it is essentially the liver alone
which takes part in the sugar-formation.

Much more important than the results of these last experiments ^ is the
above-mentioned fact that most investigators have failed to find any
evidence of glucohemia in the case of phloridzin-glucosuria. The con-
clusion has, therefore, been drawn that the elimination of sugar after
phloridzin poisoning is due to the abnormal permeability of the kidney
epithelium for sugar. Thus the sugar in the blood would become less and
less, and indirectly the liver, and perhaps the other places where glycogen
is stored, would be compelled to give up sugar. To support the assump-
tion, that the glucosuria produced by phloridzin is of a renal nature, N.
Zuntz * has published a proof, which is not, however, conclusive. He
injected, by means of a trocar, a solution of phloridzin through the walls
of the renal artery into the blood-stream. The kidney to which the glu-
coside was carried directly was the first to ehminate sugar. Pfliiger ^
believes that it is not impossible that the first separation of sugar may be
due to a decomposition of the phloridzin itself. At present we cannot be
sure as to what is the correct explanation of phloridzin glucosuria. Only
this is known, — the matter is by no means so simple as has been
assumed." Above all, it must be mentioned that starving animals will
also eliminate sugar after the introduction of phloridzin, and repeated
additions of the glucoside result in renewed glucosuria,' and to such a
degree that the question as to whether food other than carbohydrates can
give rise to the sugar formation now presents itself.

Our knowledge concerning the formation of sugar in the animal organism
was considerably increased by the discovery of von Mering and Minkowski '
in the year 1889 that dogs always suffered from severe glucosuria after
coni'plele extirpation of the pancreas. If a small part of the pancreas is left

' Inaug. Dissert. Konigsberg, 1SS7.

' hoc. cit. Z. klin. Med. 14, 415 (1888). Cf. Minkowski and Thiel: Arch. exp.
Path. Pharra. 23, 14'2 (1887).

' For a critical discussion see E. Pfliiger: Das Glykogen und seine Beziehungen zur
Zuckerkrankheit (1905).

< Arch. Anat. Physiol. 1895, 570.

* hoc. cit. p. 539.

• Cf. Carl Jakobj: Arch, exper. Path. Pharra. 35, 213 (1895).

' Cf. Lusk: Z. Biol. 42, 31 (1901). O. Loewi: Arch, exper. Path. Pharra. 47,48
(1902).

' Ibid. 26, 371 (1890). O. Minkowski: Untersuchungen iiberden Diabetes melhtus,
Leipzig, 1893.

CARBOHYDRATES.

§3

in the body, the glucosuria is either prevented or lessened. As Pfliiger '
has shown, an animal which has been wholly deprived of its pancreas
behaves outwardly quite differently from one which has a slight residue of
the gland left in the body. Pfliiger observed that after a total extirpation
tlie sugar elimination invariably appeared within the first twenty-four
hours, and lasted continuously until the death of the animal, even when it
received no further nourishment. The symptoms peculiar to a partial
extirpation, such as polydipsia (excessive thirst), polyphagia (voracity),
and polyuria (excessive urination), were either entirely lacking or barely
indicated. Usually the elimination of sugar takes place very quickly after
the extirpation of the pancreas. Thus Bierry and Gatin-Gruzewska
obtained the following results from four experiments:

Weight of
Dog.

End of Opera-
lion.

Appearance of
Dextrose.

Experiment No. 1
Experiment No. 2
Experiment No. 3
Experiment No. 4

10 kg.
14 kg.
20 kg.
14.3 kg.

1 o'clock
4 o'clock
1 o'clock
10.30 o'clock

3 o'clock
5 .35 o'clock
3.30 o'clock
3.30 o'clock

The elimination of sugar in the urine has likewise been observed after
total pancreas extirpation in Selachier,^ frogs ^ and in birds.* The results
oi)taincd after partial extirpation are not as uniform. Sometimes gluco-^^.:^
suria is observed, sometimes not. This might indicate that all the parts
of the pancreas gland are not of the same nature, so that in one operation
more of the tissue which partakes in the sugar formation is carried away
than in another. Numerous experiments by difTerent observers in this
direction have, however, established the fact that all parts of the pancreas
tend to increa.se the sugar content of the system, and thus prevent gluco-
suria. The true cause of the divergence in the results obtained by
different investigators is probably to be traced to the different methods
of operation employed.'^ Indeed, the glucosurias produced by partial and

' Pfliiger's Arch. 106, 181. Cf. Sandmeyer: Z. Biol. 31, 12 (1895). Cf. also E. W.
Pfliiger: Das Glycogen, etc., loc. cil.

' V. Diamare: Zentr. Physiol. 20, 617 (1900).

' Cf. .\ldehoff: Z. Biok 28, 29.3 (1891), and Marcu.se: Arch. Anat. Physiol. 5.39 (1894).

MV. Kausch: Arch, exper. Path. Pharm. 37, 274 (1890); 39, 219 (1897). O.
Minkowski: .\rch. exper. Path. Pharm. 31, 85 (189.3). Cf. v. Diamare: Zentr.
Phy.siol. 19, .545 (1905). Cremer .and Ritter: Z. Biol. 28, 4.59 (1891).

* Cf. De Renzi and Reale: Berliner khn. Wochenschr. No. 23 (1892). J. Thiroloix:
Diabete-pancr<5ati(iue, p. 95 (1892). von Mering and Minkowski: Diabetes mellitiis
nach Pancreasexstirpation, p. 12 (Leipsic, 1899). W. Sandmeyer: Z. Biol. 31, 74 and
85 (1894). E. HC-don: Travaux de physiologie, 1-150, Paris, 1898.

84 LECTURE V.

total extirpation of the gland are very similar, and only show gradual
differentiations. .Also, it must not be forgotten that the pancreatic
gland, like other organs, is in its entire function without question
dependent upon nervous influences, and that under certain conditions
disturbances and injuries in the region of the nerves which are connected
with the pancreas may produce glucosuria. The fact that the operation
of itself does not cause elimination of sugar, has often been shown, neither
does the extirpation of the solar plexus, or at least not permanently.
Before we take up the explanation of this disturbance in the metabolism
of carbohydrates produced by extirpation of the pancreas, it may be well
to describe briefly the phenomena exhibited by the organism after it has
been deprived of the gland. In general, dogs do not survive the operation
very long; at best they can be kept alive only two or three weeks. Pfliiger
found the cause of death to be extensive suppuration. According to
him, it was not the lack of the gland, nor the glucosuria produced, which
caused the death, but rather that the wound did not heal on account of
the sugar in the tissues. That this view is correct is shown by the fact
that if a piece of the pancreatic gland is left in the abdominal cavity, gluco-
suria ensues only after this piece is dead, and such dogs live much longer.
By dissection of such a dogT, Pfliiger ' found an extensive atrophy. The
individual organs did not show much signs of disease. The fatty tissue
had disappeared. The liver showed a remarkable condition. All the other
organs, except the brain, heart, and kidneys, which retain their weight
even during inanition, had lost considerably in weight; the liver, on the
contrary, had increased. Its weight represented 4.77 per cent of the body
weight. Normally, the liver, according to Pavy,^ amounts to from 3 to
4.7 per cent of the body weight, and after 28 days of starvation the
value falls to 1.5 per cent. The composition of the liver was found to be
as follows:

Per cent.

Dry substance in the fresh liver 24.2

Fat in the fresh liver 2.7

Fat in the dry substance 11.2

Water in the fresh liver after extraction of the fat ... 78 . 3

Dry substance in the fresh liver after extraction of the fat . 21.7

Nitrogen in the fresh liver 3.2

Nitrogen in the dry liver 13.2

Nitrogen in the dry Uver after extraction of the fat . . . 14.9

' Pfliiger's Archiv. 108, 115 (1905).

' Phil. Trans, for 1860, p. 579. Researches on the Nature and Treatment of Diabetes,
London, 1862.

CARBOHYDRATES. 85

The liver contained 0.0259 gram of glycogen. This shows that it hail
retained the power of forming glycogen.

Pfliiger also examined the muscles, and, as in the case of the liver, found
values which were not different from the normal. The only striking fact
was the high percentage of water. In spite of this agreement of com-
position in the case of the liver and muscles (which obviously play the most
important part in the metabolism of carbohydrates) with the values
obtained under normal conditions, it is on no account permissible to draw
the conclusion that as a matter of fact there has been no change in the
materials which go to make up these tissues. As we shall see subse-
quently,our methods of analysis arenot sensitive enough, and our knowledge
of the cell-constituents is far too inadequate for us to attempt to answer
such questions with exactness. It is of importance, first of all, to know-
that so far as we are now able to judge, the liver, like the organs indis-
pensable to Ufe [e.g., the brain, heart, and kidneys], is protected during
inanition at the cost of all other tissues. From this the conclusion may
be drawn that evidently the liver is not simply cut off from the metabolism
of carbohydrates during the whole duration of glucosuria, but, on the
contrary, is continually acting vigorously. It gives up the large amounts
of sugar which are found in the urine, and in it evidently takes place, as we
shall see, the transformation into sugar of substances not belonging to the
carbohydrate group.

The fact that the sugar content of the blood rises after total extirpation
of the pancreas, while at the same time the glycogen content of the organs,
the liver especially, remains low, is a matter of considerable importance.
A true glucohemia naturally ensues which causes glucosuria. Thus the
cause is the same as in all the other cases of sugar elimination, w^hich have
been observed up to the present time, phloridzin glucosuria po.ssibly
forming an exception. We are now ready to take up the question as to what
causes the glucohemia. It is a matter of fact that it appears as soon
as the pancreatic gland is removed, which suggests the thought that the
I0.SS of the function of this gland is the cause of the observed disturbance.
First of all we must remember that the pancreas plays an important part
in the digestion taking place in the alimentary canal. We have seen that
the breaking down of starch in the bowels is brought about principally by
the action of the diastase from the pancreatic gland. On the other hand,
it is certain that on taking away the ferments of the pancreas the alisorption
and assimilation of all the remaining foods, and consequently the whole
metabolism, must suffer. We have then to decide, first of all, whether the
glucosuria produced by the removal of the pancreas can be traced to the
absence of the digestive ferments. This must be answered in the nega-
tive, because, for one thing, if the ducts of the pancreas are Hgated,
glucohemia does not develop. Then again the greater part of the

86 LECTURE V.

gland may be removed, and, in fact, that portion directly connected
with the intestine, and glucosuria does not take place as long as a small
part of the tissue of the gland still remains in the body. A further proof
that pancreatic-glucosuria is not intimately connected with digestion is
shown by the fact that in the case of total extirpation of the gland, sugar
still appears in the urine after prolonged starvation, i.e., after the stomach
and intestines have become empty. Furthermore, it is not possible to
influence the existing glucosuria by feeding the gland to the animal. It is
interesting that after a partial elimination of the pancreas the assimilation
limit for sugar is considerably decreased. This is shown by the fact that
an animal experimented upon which did not show glucosuria would
eliminate sugar in the urine when the amount of carbohydrates fed to it
was increased, especially if the food contained glucose.

It is particularly significant that a small piece of the gland tissue
usually suffices to keep the entire metabolism of carbohydrates in nor-
mal paths. This was shown very strikingly by the experiments of
Minkowski, which proved conclusively that it was not the severe operation
itself, but the total removal of the pancreas function, that caused the
great disturbance in the metabolism of carbohydrates. In dogs the lowest
part of the descending branch of the pancreas does not grow together with
the duodenum, but lies free in the mesentery. Minkowski separated
this piece from the remaining tissue of the pancreas so that it was still in
connection with the mesentery and without disturbing its supply of blood
and lymph vessels. This piece of pancreas was then drawn out through
the abdominal cavity, grafted under the skin, and allowed to heal to-
gether with the wound. After the animal had survived this operation,
the abdomen was again opened, and all of the rest of the pancreas removed
from the body. Thus only the small portion of the gland which had been
transplanted under the skin remained in the animal, and yet glucosuria did
not ensue. If, however, this part of the pancreas was finally removed,
sugar appeared in the urine at once.

The fact that, in many cases, a small portion of the pancreas left in the
body serves to prevent the appearance of glucohemia, suggests to us
other possibilities. It is perfectly conceivable that the pancreatic gland
serves, like the liver, for example, to neutralize injurious substances, and
especially those which interfere with the metabolism of carbohydrates.
When the pancreas is removed, these products perhaps pass unhindered
into the circulation, and prevent the normal breaking down of sugar. If
this view were correct, it would follow that the injection of the blood from
an animal suffering from glucosuria into the circulation of a healthy
animal would inevitably produce the same disease. That this is not the
case was shown by the direct experiments of Minkowski and von Mering.

The only remaining explanation of pancreatic-glucosuria would seem to

CARBOHYDRATES. 87

lie in the assumption that the pancreatic gland produces a substance which
either directly or indirectlj- influences the metabolism of carbohydrates.
It is altogether out of the question to imagine that the sugar in the blood
undergoes any change by passing through the gland. The regulation of
the carbohydrate metabolism must take place indirectly, i.e., the tissue of
the pancreas influences in some way or other the organs whose task it is
to build up or to consume the sugar. This assumption has gradually
gained ground, especially after the experimental explanation of Lepine
concerning pancreatic-glucosuria had been found untenable. Lepine,' as
has been mentioned, discovered the presence of a ferment in the blood
which was able to decompose sugar. This glucolytic ferment was assumed
to be produced by the pancreatic gland. The ferment was supposed to pass
continually through the thoracic duct into the blood and circulate in this
attached to the white corpuscles. Lepine found that in animals deprived
of the pancreas there was a considerable diminution of the amount of this
ferment, or, in other words, the power of the blood to consume sugar
was considerably diminished. Thus the assumption was made that pan-
creatic-glucosuria was a result of the fact that the ferment was not formed
after the pancreas was removed. Lepine's views, however, were soon
contradicted, and to-day the hypothesis may be said to be completely
shattered. De Dominicis,^ for one, showed that in animals suffering from
pancreatic-glucosuria there was no diminution in the elimination of sugar
when blood from the portal vein of normal animals, which according to
Li'pine would be rich in the ferment, was injected into them. On the
contrary, the result was that the glucosuria became more severe. Arthus '
found that the blood contained in ligated vessels was not capable of
decomposing sugar, and therefore contained no glucolytic ferment. More-
over, from many sides it has been shown that glucolysis appears as a post-
mortem phenomenon, and that it has any connection with the metabolism
of carbohydrates in the living organism has been flatly denied. In fact, the
whole theory of glucolysis in the blood rests upon an extremely slight
foundation, and a great deal maybe said against it. Lepine in his experi-
ment failed entirely to take into consideration many phenomena which
appear after extirpation of the pancreas, such as, for example, the fact that
after the operation the liver loses its glycogen. In this connection,
Marcuse's * observation is interesting that glucosuria in frogs, resulting
from extirpation of the liver, disappeared if at the same time the pancreas
was removed.

In the last lecture, we found that the muscles are the chief consumers

• Compt. rend. 113, 729 (1891); ibid. 113, 1014 (1891).

' Wiener med. Wochschr. 42—15 (1898).

5 Arcli. Physiol. 1891, 42.5; 1892, .3.37.

' Z. klin. Med. 26, 225 (1894). Cf. .\. Montuori: .Vrch. ital. biol. 25 (1896).

88 LECTURE V.

of sugar. Their necessary supply of carbohydrates is regulated by the
liver. It would seem possible, therefore, that the function of the liver
might be disturbed in some way by the removal of the pancreas. The
hver stores up the resorbed sugar in the form of glycogen. It might seem
possible that the liver in an animal deprived of the pancreas is no longer
able to retain the resorbed sugar, i.e., withdraw it from the general metab-
olism, so that in this way the blood is flooded with sugar. Now Pfliiger, as
we have seen, showed that the liver, even after long-continued glucosuria, is
still capable of forming glycogen. At least the ability is not entirely want-
ing. Furthermore, this assumption does not explain the fact that starving
animals also exhibit glucohemia. This latter fact suggests to us another
way in which the liver-function may be disturbed, namely, with regard to
the decomposition of glycogen. Hand in hand with the consumption of
sugar in the muscles, there takes place the transformation of the stored-
up glycogen into sugar. An extremely fine regulating mechanism pre-
vents large amounts of glycogen being suddenly decomposed in such a
way that the blood would be flooded with sugar. We have already met
with this regulation in discussing the glucosuria produced by the " diabetic
puncture," and the elimination of sugar in the urine caused by the injection
of salt solution into the circulation. We saw at that time how evident it
was that nervous influences had a great deal to do with keeping the glycogen
conditionof the liver along definitepaths. What is unexplained is merely how
the breaking down of the glycogen takes place. It is a result of diastatic
action. It is inexplicable why the diastase, which is evidently present in
the liver, at one time attacks the glycogen and at another leaves it alone,
unless we assume that either the glycogen is present in a condition such
that it cannot be acted upon by the ferment, i.e., is in some form of loose
chemical combination, or that the diastase becomes active only at the
time it is required.^ We are acquainted with many ferments, as we shall
eventually see, which are secreted by the cells in an inactive condition.
In such cases the presence of another substance usually formed by
an entirely different kind of cells is necessary in order to make the
ferment " active." Such processes have not been sufficiently studied
in the case of diastase. We may assume, however, that by union with
some sort of substance, perhaps the protoplasm of the cells, the diastase
is inactive and becomes free only at such a time when it is needed. At

' The assumption that the breaking down and building up of glycogen takes place
in the same way that, for example, a hydrolysis or a synthesis may be brought about
artificially by the action of one and the same ferment according to the concentration
ratio (cf. p. 38) is not well established at present, for the products formed artificially
are not those expected, but their isomers ; and furthermore, it is not known that the living
cell contains the condition established in chemical experiments. Cf. Hofmeister's Die
Chemische Organisation des Zelle. Viewig and Sohn, Braunschweig, 1901.

CARBOHYDRATES. 89

present our knowledge in this direction is still too slight for us to
attempt to discuss in the light of experimental data any disturbance
in the glycogen decomposition. We merely wish to point out the possi-
bilitj', and to suggest that there is a certain analogy between pancreatic-
glucosuria and the disturbances in the formation of sugar which have
been taken up prcvioush^ (diabetic puncture, etc.).

Glucohemia might also become established by the failure of the
muscles to consume sugar. Unfortunately we know very little con-
cerning the manner in which the muscles consume sugar, and conse-
quently practically nothing concerning the possibilities of disturbing
this function. In analogy to other processes, it has been suggested that
this may also be due to the action of a ferment, a conception which is per-
fectly plausible, for we have here to deal with either a direct or indirect
protoplasmic action. Recently 0. Cohnheim ' has studied this problem.
He showed that the juice obtained from the pancreatic gland by high
pressure was not capable of decomposing sugar. On the other hand,
sugar was not attacked by the expressed juice from the muscles. Cohn-
heim found, however, that on bringing together the juices from both of
these organs, glucolysis took place at once. He explained this fact by
considering the analogy with observations made with other ferments, and
assuming that the muscles produce a ferment which is inactive, i.e., inca-
pable by itself of attacking sugar. This muscular ferment is activated by
a suixstance obtained from the pancreatic gland and brought to it by the
blood circulation. This would readily explain pancreatic-glucosuria. It
must be remembered, however, that this conception does not explain all
of the phenomena observed in pancreatic-glucosuria; thus, for example,
it does not account at all for the fact that the glycogen stores of the liver
disappear. On the other hand, there is absolutely no reason for assuming
that the pancreas has only one function with regard to the metabolism of
carbohydrates. It is, indeed, possible that it has different effects upon
different organs, and that, furthermore, when the functions and metabolic
effect of the different organs become disturbed, they again bring into play
secondary influences of the organs upon one another, so that one disturb-
ance causes a number of complications.

If we summarize all that we know positively concerning the cause of the
glucohemia resulting from extirpation of the pancreas, we may say that
we have to deal with a disturbance in the regulation of the transforma-
tion of sugar, and that evidently the pancreatic gland gives up to the blood
some substance which regulates the metabolism of carbohj'drates. This
function of the pancreas, in contrast to its other function of forming and

' Z. physiol. Chem. 39, 336 (1903); 42, 401 (1904); 43, 547 (1905). For objections
to Cohnheim's conclusions, see Claus and EmbJcn: Pankreus und Glycolyse, Ilofineister's
Beitrage, 6, 214, 343 (1905).

90 LECTURE V.

secreting the digestive ferment, is spoken of as that of an internal secretion.
An internal secretion is something formed within a glandular organ and
given off to the blood or lymph. The ordinary pancreatic juice is called
an external secretion.

The discovery by Langerhans ' of a peculiar segregation of cells in the
pancreatic gland led to discussion as to whether the gland possesses partic-
ular cells for its various functions. These cell-forms — called islands of Lan-
gerhans — which stand out very sharply from the other cells in the gland
differ from the latter not only in outward appearance, but, by the fact that,
unlike the ordinary secretory cells, they have no connection with the exit
ducts from the gland. More recently Diamare and Kuliabko ^ have taken
up anew the question as to the significance of these cells. They studied
the pancreatic glands of the Teleostei because in these animals the islands of
Langerhans are relatively large, and preparations of them may be easily
made free from the other cells of the pancreas tissue. They found that
only the ordinary cells of the gland produced an amylolytic ferment,
while the cells of the islands of Langerhans possessed the power of destroy-
ing dextrose. This sums up all that we know concerning these islands of
Langerhans, and it remains undecided as to whether they form an internal
secretion or not. We shall come back to this point in the discussion of
diabetes.

Changes in the pancreatic gland had been observed before the discovery
of pancreatic glucosuria, namely in the so-called diabetes mellitus of
man. Although, in discussing the phenomena of this pathological degen-
eration, we are leaving the proper field of physiological chemistry, we will,
nevertheless, take it up more or less in detail, because in this disease we
have in a certain sense an experiment brought about by Nature, which
serves to give us some insight into the normal metabolism of carbohy-
drates. We shall, however, discuss the disease only in so far as it is directly
or indirectly connected with the metabolism of carbohydrates, and leave
the discussion of the remaining clinical symptoms of this very interesting
disease to the text-books on clinical medicine.

Diabetes has been known for a long time.^ The Indian and Arabian
physician of the Middle Ages recognized the fact that associated with the
disease was the elimination of a sweet substance in the urine. It remained
for Th^nard, in 1806, to isolate this sweet substance; Chevreul crystallized

' Beitriige zur mikroskopischen Anatomie der Bauchspeicheldriisen, Berlin.
Dissert. 1869.

' Zentr. Physiol. 18, 432 (1904).

' Cf . Max Salomon : Geschichte der Glukosurie von Hippokrates bis zum Anfang des
19 Jahrhunderts, Deutsche.s Arch. klin. Med. 8, 489 (1871'), and E. O. v. Lippmann:
Zur Geschichte des diabetischen Zucker. Chem.-Ztg. 29, 1197 (1905).

CARBOHYDRATES. 91

it in 1S15; while Bouchardat ' and Peligot^ succeeded in identifying it as
dextrose, or grape-sygar, in 1838.

The cause of this disease has for a long time been attributed to a marked
glucohemia. This naturally does not account for the whole nature of the
disease, but is only one of many symptoms. It may be caused in a number
of different ways; and, according to all we know at present regarding
diabetes, there is no longer any doubt that diabetes does not represent
a single disease, but rather that the glucohemia, or rather the resulting
glucosuria, is merely a symptom most readily recognized, and is produced
by the most varied pathological conditions. For this reason it would be
out of the question to attempt to find a common cause of glucohemia.
The disturbance in the metabolism of the carbohydrates varies in different
cases.

We distinguish between a light and severe form of diabetes. In some
cases sugar is eliminated in the urine only after the patient has partaken
of starch or of glucose. In such cases there is no noticeable glucosuria
if the diet is restricted to meat and fats. These mild forms show all
stages of alimentary glucosuria, and make it seem probable that the limit
of an assimilation for carbohydrates has been considerably diminished.
In many cases sugar is found in the urine only when carbohydrates are
eaten on an empty stomach. Often muscular work suffices to arrest the
sugar elimination. In other cases, the glucosuria lasts only while the
absorption of sugar continues in the intestine. The cause of this kind of
diabetes is generally attributed to a weakening of the liver function. The
latter is obviously not able to work over the sugar quickly enough into
glycogen. It permits too much sugar to get into the circulation. Thus
a glucohemia results, which after a time is compensated by the elimination
of sugar by the kidneys, only to appear again from the same cause as before.
Against this assumption the objection has been raised that the liver may
suffer most severe changes, without the appearance of sugar in the urine.
This is, however, not a serious objection, for we know that the liver has
qiute a number of different functions, of which each is to a certain extent
independent of the others, so that one function by itself may be disturbed.
It does not follow that every disease of the liver will attack that part of
the cells which participates in the regulation of carbohydrate metabolism.
A general weakening of the activity of the liver cells can cause diabetes;
thus it is met with in persons of undermined constitution. Possibly the
fact established by Hofmeister ' that dogs, after all forms of nourishment
had been withheld for a considerable time, eliminated sugar in the urine,

• Compt. rend. 6, 337 (1838).

> Ibid. 7, 106 (18.38).

' Arch. exp. Path Pharmak. 26, 355 (1890).

92 LECTURE V.

shows that many forms of glucosuria belong in this category. It is not
impossible, but on the contrary extremely probable that many of these
light forms of diabetes result from conditions similar to those resulting
from the diabetic puncture, etc. The only difference here is probably
that in the case of the operation we have but a single shock to the system,
whereas here there is evidently a permanent irritation of the sugar center.
This coincides with the fact that many persons afflicted with the disease
are very nervous.

Between these light forms of diabetes and the more severe types, there
are, as we have said, all stages of intermediate types, and not infrequently
the former change into the latter. In the first case the disease is not of
a very bad character, and appears chiefly in elderly people, producing
symptoms which are easily understood, but in the more serious types we
are astonished at the severity of the disease. How great the disturb-
ance in the carbohydrate metabolism is, may be illustrated by the fact
that the elimination of sugar continues even after carbohydrates are
entirely withheld from the diet, and the patient eats, for example, only
meat and fat.

Let us now turn our attention to the main symptom of diabetes, the
glucohemia. Why does this exist? There are a priori two possibil-
ities. On the one hand, the amount of sugar formed may be abnormally
large; or, on the other hand, the sugar produced normally may not be
consumed as it should be, and thus lies unutilized in the tissues only to be
removed finally from the organism as a waste-product. The last e.xpla-
nation is really the more plausible; for although we can easily conceive
that there might be temporarily an increased formation of sugar, possibly
from fat or perhaps from albumin, it is not easy for us to understand
how this could take place continuously. Furthermore, the whole behavior
of the patient does not correspond to any such assumption. Fats and
albumin agree with him. With their help and the removal of carbo-
hydrates from the diet, it is possible greatly to diminish the elimination of
sugar. On the other hand, the glucosuria immediately becomes more
severe if carbohydrates are fed to the sick. In these severe cases we cannot
account for the facts by assuming that the liver has lost its power of storing
up sugar in the form of glycogen. The disease continues during a period
of fasting, long after all carbohydrates have left the alimentary canal.

The fact that the liver actually retains its abihty of storing up sugar in
the form of glycogen is proved by the fact that glycogen has been re-
peatedly found in the livers of those who have suffered from severe
diabetes.* This, however, is not always the case. Sometimes the liver

' Cf. Kuhne: Virchow's Arch. 32, 536 (1865). Jaffd: ibid. 36, 20 (1866). Kiilz:
PflOger's Arch. 13, 267 (1876). J. v. Mering: ibid. 14, 274 (1877). Atieles: Zentr.
med. WiBsensch. 23, 449 (1885). F. Th. Frerichs: Ueber den Diabetes, Beriin, 1884.

CARBOHYDRATES. 93

contains glycogen, and sometimes it does not contain a trace of this poly-
saccharide. This is not astonishing when we remember that the name
diabetes includes quite a number of different diseases, all of which show
the common symptom of glucohemia.

We will now attempt to answer the question as to whether diabetes can
be explained entirely on the assumption that it is due to a diseased pancreas.
It is certain!}' interesting to compare diabetes with the glucosuria pro-
duced by extirpation of the pancreas. It has been shown in many cases
that the pancreas of diabetics was diseased, and there is absolutely no
doubt but that there are forms of diabetes which originate in a disturbance
of the functions of the pancreatic gland. Recently changes have been
noticed, particularly at the islands of Langerhans, and also many degen-
erations have been observed, e.g., hyaline degeneration. At the present
time it is impossible to decide whether the conclusion may be drawn that
there is positively a connection between the cells of the Langerhans group
and the metabolism of carbohydrates. At all events, the fact that in many
cases of diabetes the post-mortem examination shows these cells to be
absolutely normal, does not necessarily show that any such assumption is
false, for it is not possible to trace all forms of diabetes back to a common
cause; and furthermore, the fact that there is no noticeable histological sign
of a change having taken place in the tissues, does not prove that the cells
have suffered nothing as regards their functional activity. Perhaps the
researches of Diamare and Kuliabko ' will eventually settle this question.

Let us return to the question as to the sugar formed not being consumed.
It is possible that diabetics in general have a limited capacity for oxida-
tion. On the other hand, the fact that the other forms of nourishment are
taken care of normally speaks a priori against any such assumption. The
following experiments are a direct proof that the organism of diabetics
possesses its full oxidation capacity. O. Schultzen^ found that diabetics
readily consume the alkali salts of lactic and the vegetable acids, also
inosit and mannitol. M. Nencki and N. Sieber ^ observed that patients
suffering from severe diabetes could take care of the difficultly-oxidizable
benzene just as well as the healthy organism could. Direct respiration
experiments likewise showed that the respiratory exchange in such cases
— severe cases of diabetes — did not var}' from the proper physiological
relations. It is true that von Pettenkofer and C. Voit * found that there
was a considerable diminution in the inspired oxygen and expired carbon
dioxide, and drew the conclusion that this was due to a decreased capacity
for oxidation; but this conclusion was later abandoned by Voit ' himself,

Loc. cit. ' Ber. klin. Wochschr. No. 35 (1875).

J. pr. Chem. N. F. 26, .35 (18S2). ' Z. Biol. 3, .380 (1867).

Physiol, allg. Stoffwechsels und der Erniihrung (1881), p. 227, et aeq.

94

LECTURE V.

who found that the limited absorption of oxygen was a result of the dis-
turbed carl3oh3'drate metabolism, rather than the cause of it. The absorp-
tion of oxygen depends upon the combustion taking place in the body.
Leo,' as well as Weintraud and Laves,- has finally shown that the amount
of absorbed oxygen is the same with healthy people that it is with those
suffering from diabetes of equal weight and condition of nourishment, and
finally that the apparent decrease in the elimination of carbonic acid is
caused by the faulty decomposition of carbohydrates.

Of especial importance as regards the cause of the insufficient consump-
tion of the sugar formed in the organism are the experiments performed by
0. Baumgarten ^ under the direction of von Mering. Baumgarten, at von
Mering's suggestion, tried some feeding experiments upon diabetics, and
upon dogs with removed pancreas, employing substances which are re-
garded as decomposition or oxidation products of the sugars. It was
found that d-gluconic acid, d-saccharic acid, mucic acid, glucuronic acid,
glucosamine-hydrochloride, succinic acid, d-tartaric acid, saUcylic alde-
hyde and vanillin, were consumed by diabetics just as readily as by
healthy individuals. The following summary illustrates the relation
between some of these products and rf-glucose (also called dextrose, or
grape-sugar) :

CHO COOH CHO COOH COOH

I I I I I

H— C— OH H— C— OH H— C— OH H— C— OH H— C— OH

1 I I I I

HO— C— H HO— C— H HO— C— H HO— C— H HO— C— H

I I I I I

H— C— OH H— C— OH H— C-OH H— C— OH HO— C— H

I I I I I

H— C— OH H— C— OH H— C— OH H— C— OH H— C— OH

CH20H

CH2OH

COOH

COOH

COOH

d- gluco.se

c?-gluconic

d-glucuronic

d-saccharic

d-mucic

acid

acid

acid

acid

These experiments show that the organism of diabetics can decompose
in the same way as the healthy organism, substances which in their alde-
hydic nature are closely related to dextrose, and are to be regarded as
direct oxidation products of dextrose; in other words, a very slight
oxidation of the sugar molecule suffices to enable the organism of the
diabetic to attack it. In this way the views expressed by Scheremet-
jewski,^ Schultzen,'' A. Cantani," and of Nencki and Sieber gain ground;

' Z. klin. Med. 19 (1890). ' Z. physiol. Chem. 19, 603 (1894) ; 19, 629 (1894).

' Z. exper. Path. u. Therapie, 2, 53 (1905).

' Arbeiten aus dem physiol. Institut zu Leipzig, 1868.

' Loc. cit. ' Du Diabetes mellitus, Berlin, 1877.

CARBOHYDRATES. 95

and, as a matter of fact, it seems probable that the principal cause of
glucohemia and the resulting glucosuria is that the organism has lost the
power of splitting the sugar molecule. The complete combustion of the
glucose molecule is, according to this view, always dependent upon a
previous hydrolysis, or some attack which loosens up the sugar molecule
and disturbs the condition of equilibrium; without some such action the
combustion cannot take place in the organism. This assumption is sub-
stantiated by the fact that glucose is very readily consumed b\' the healthy
organism, even more readily than is the case with other foods. Thus in
phosphorus poisoning, the oxidizing power is lost to a considerable extent
without the appearance of glucosuria.

We have again come back to the undecided question concerning the
normal breaking down of sugar. The answer depends upon the explana-
tion of the inability of diabetics to break up glucose. It is easy to con-
ceive that the principal places of sugar consumption, the muscles, produce
a ferment which serves first of all to loosen up the glucose molecule and
thus prepare it for oxidation. That the glucose is not itself directly
oxidized is very plausible. In this way, the sugar stores in the organism
are in a measure protected. Only at the moment of the expenditure of
energy do the muscular cells prepare the dextrose molecule for consump-
tion. As to why the diabetic has lost this power, whether the ferment
which starts the process is missing, or whether he has lost the power of
activating this ferment, are questions which cannot be answered positively
at the present time. An important observation has been made that when
cane-sugar is taken into the system often only one half of the molecule
appears subsequently in the urine. This is due to the fact that many
diabetics are able to oxidize and assimilate fructose without difficulty.'
In fact, because the liver can in such cases take care of fructose and not of
glucose, it has been proposed to feed diabetics a carbohydrate, such as
inulin, which on being hydrolysed yields fructose rather than glucose.
Unfortunately, however, the above carbohydrate is so difficult I3' digestible,
that the experiment has not met with much success.^ At all events, it is
most remarkable that the liver should transfer fructose into glycogen,
a process which according to the generally accepted opinion involves the
intermediate formation of glucose; and yet should not be able to use the
glucose which it receives as such.

' E. Kiilz: Beitrage zur Pathologie und Therapie des Diabetes raellitus, p. 130, Mar-
burg, 1874. Worm-Muller: Pfliiger's Arch. 34. 576 (1884); 36, 172 (1885). F. Hof-
meister: Arch, expcr. Path. Pharm. 26, 240 (1889). J. Haycraft: Z. physiol. Chem.
19, 137 (1S94), Minkowski: Arch, exper. Path. Pharm. 31, 158 (1898). Sandmeyer:
Z. Biol. 31, 12 (31) (1894). Fr. Voit: Z. Biol. 29, 147 (1892). Socin: Wie verhalten
sich Diabetiker Liivulose- und Milchzuckerfiitterung gegeniiber? Dissert. Strassburg,
1894.

' Sandmeyer: loc. cit. and Miura: Z. Biol. 32, 279 (1895).

96 LECTURE V.

The assumption that in some way or other the muscles have lost the
power of preparing sugar for consumption does not explain the cause of
diabetes, for the real beginning of the chain of disturbances which con-
stantly brings into play new effects is not, it is certain, to be attributed
originally to a faulty combustion of sugar; in fact, the combustion of
sugar is not entirely lost in the organism of diabetics. We come back
here, as in the case of most disturbances in metabolism, to the individual
cells themselves and their dependence upon the nervous system. Even
if we attribute the breaking down and formation of glucose entirely to
the action of ferments, we fail even then to get around the responsibility of
the cells, for it is they which form the ferment, and the production of the
latter seems in many cases to be dependent upon nervous influences.
Now if, furthermore, we add to this the fact that many ferments are
known which are secreted by the cells in an inactive condition, and are
only activated by means of a second ferment produced by other cells — •
often those of another «rgan — then it becomes easy for us to understand
how disturbances may originate at countless places in the w'hole mechanism.
These all together may accomplish the final effect, namely, a glucohemia
in consequence of the non-consumption of a part at least of the glucose,
whether on account of a disturbance in the nervous system, or the fact that
the activating agent (perhaps furnished by the pancreas) is lacking, or
whether it may be because the muscular cells themselves are diseased.
In discussing these possibilities, it must be brought forward once again
that in glucohemia we have merely a symptom, a consequence and not a
cause, which without question, by means of the resulting changes in the
composition of the tissue and of the blood, may effect all sorts of secondary
disturbances, and which again, by a continuous circulus vitiosus, tends to
diminish the life energy of the body cells and aggravate more and more
the nature of the disease. In investigating the causes of pathological
phenomena, the endeavor must be more and more to study the purely
functional disturbances on the basis of biological experiments, for doubt-
less such may be present without there being any indication of mor-
phological changes discernible by present methods of-investigation.

We have learned to consider glucosuria as an essential symptom of all
forms of diabetes. It can attain quite remarkable proportions: — in a
single day as much as a kilogram (2.2 pounds) of sugar may be eliminated.
Besides glucose we occasionally meet with an elimination of other kinds
of sugar; for example, fructose and certain pentoses. Furthermore, in
the urine of diabetics, often higher molecular sugars, such as maltose and
dextrin-like compounds, have been detected without our being able to
draw any conclusions from the occurrence of these products of an evidently
incomplete combustion, as to the nature of the disturbance in the metab-
olism of carbohydrates. In severe cases of diabetes the urine has a

CARBOHYDRATES. 97

peculiar, fruity odor which was noticed by the older physicians. This is
due, as C. Gerhardt ' supposed and v, Jaksch^ has shown, to the presence
of acetoacetic acid (CH3CO)CH2 . COOH. Besides this compound acetone
CH3 . CO . CH3 and, as Minliowski ^ has shown, /9-hydro.\y-butyric acid
CH3CH(OH)CH2 .COOH, are also present in many cases. All three of
these compounds, as a glance at their structural formula? will show, stand
in direct relation to one another. Acetoacetic acid is evidently formed
by the oxidation of ,3-hydroxy-butyric acid:

* CH3.CH(0H) . CH2 . COOH + O = (CH3 . CO) CH, . COOH + H2O
^-hydroxy-butyric acid acetoacetic acid

From acetoacetic acid, acetone is readily formed by loss of carbon
dioxide :

(CH3 . C0)CH2 . COOH = CH3 . CO . CH3 + COo
acetoacetic acid acetone

The fact that in many cases j3-hydroxy-butyric acid is not found in the
urine when the other two compounds are present, is not remarkable, for
we can easily understand that in one case the former is oxidized and in
another case is not. ,9-hydroxy-butyric acid contains, as the al)ove formula
indicates (*), an asymmetric carbon atom, — i.e., it is optically active; as
a matter of fact, the left-i;otating form is always eliminated.

It has been attempted repeatedly to establish the origin of these com-
pounds in the urine, and especially their significance as regards disease.
It is above all worth mentioning that the occurrence of acetone in the
urine (acetonuria) is not peculiar to diabetes. To some extent it has been
observed in the urine of many fever patients. Acetone and acetoacetic
acid have also Ijeen found after long-continued inanition.* Acetone is
also eliminated in small amounts when healthy individuals are fed exclu-
sively with alliumin antl fat. All of the acetone does not leave the system
through the kidneys, but a part is given off by the lungs. The question as
to the origin of these so-called acetone bodies ^ has been the subject of
considerable controversy. The simplest explanation is that they are in
some way directly connected with the disturbance in the metabolism of
carbohydrates," for the observation that acetonuria occurs after inanition

' Wiener med. Presse. 28, 67.3 (1SG5). Cf. Fetters Prager Vierteljahresschrift, 55,
81 (1857). Jaksch: Z. physiol. Chem. 7, 485 (188.3).

' Ber. 15, 140() (1882).

' Zcntr. med. Wiss. 212 (1884). Kulz: Arch, expcr. Path, Pharm. 18, 291 (1SS4);
Z. Biol. 20, 105 (1884); 23, 329 (1887). O. Minkowski: .\roh. e.'ciier. Path. Pharm.
31, 183 (IS93). Araki: Z. physiol. Chem. 18, 1 (1894).

' von Jaksch: Ueber Azetonurie und Diazeturie, Berlin, 1895. Fr. Muller: Berlin,
klin. Wochschr. 428 (1887).

' H. C. Geelmuyden: Z. physiol. Chem. 23, 431 (1897).

' Cf. F. Maignon: Compt. rend. 140, 1124 (1905).

98 LECTURE V.

does not disagree with Hofmeister's ' experiment showing that glucosuria
is produced in starving dogs. It has been proved, however, that an
existing acetonuria is diminished and even stopped by Umiting the supply
of carbohydrates, while, on the other hand, it is a matter of experience
that when carbohydrates are entirely removed from the diet of diabetics
the direct consequence is often an acetonuria. For this reason it has been
attempted to ascribe the appearance of these acetone bodies to other
sources, to albumin especially. The appearance of the acetone bodies is,
according to one view, due to the progressive decomposition of albumin
taking place in severe types of diabetes. Now there is no parallel between
the elimination of nitrogen and acetone bodies. In starvation experi-
ments, for example, on account of the lessened consumption of albumin
during the first days, the amount of acetone eliminated increases.^ Ac-
cording to Weintraud,' diabetics can be in equilibrium as regards nitrogen,
or may even add to their nitrogen without the elimination of acetone being
affected in the slightest. The observation made by Magnus-Levy * is like-
wise contrary to the assumption that the acetone bodies result from albumin ;
in a case of diabetes there were 262 grams of albumin decomposed, and 342
grams of /9-hydroxy-butyric acid eliminated. This does not show that ace-
tone cannot be formed from albumin, for there may be more than a single
source; and then again the discussion of this cjuestion brings us once more
to the possibility of one food-stuff being converted into another. Further-
more, it must be admitted that our present knowledge concerning the
intermediate decomposition of albumin is still very limited. In no case
does it follow necessarily that the amount of nitrogen and of sulphur
eliminated is to be taken as a measure for the total decomposition of the
albumin introduced into the body. Again the point may well be raised
that the carbon eliminated in the urine represents only a part of the
carbon contained in the albumin, and the remaining carbon can be retained
in the system for a consideraI)le time after all of the nitrogen and sulphur
have been eliminated.^ Again there is much to be said in favor of fat
being the mother-substance of the eliminated acetone. This agrees with
the fact that during starvation for a time the organism performs its tasks
at the expense of its own fat, while, at the same time, the elimination of
acetone increases. The fact that feeding carbohydrates lessens the ace-

' Loc. cit.

' Cf. Giuseppe Satta: Hofmeister's Beitrage, 6, 1 (21), and 6, 376 (1904).

» Arch, exper. Path. Pharni. 34, 169 (1894).

' Ibid. 42, 149 (1899), and 45, .389 (1902).

' Acetone has been obtained in the laboratory to a sHght extent by the oxidation of
albumin. These experiments, however, have no relation to the formation of acetone
in the body. The amount formed is far too small, and the possible source in fats or
carbohydrate is not excluded.

CARBOHYDRATES. 99

tonuria is explained by a decreased decomposition of fat. If large quan-
tities of albumin are fed to the organism, the elimination of the acetone
bodies is likewise abated. Conversel}', if the diet is made to consist of
fat with exclusion of carbohydrates, more acetone is formed. Schwarz ^
observed, moreover, that the blood of diabetics always contains an exces-
sive amount of fat.

For the present, we must leave the question of the source, or, perhaps
more correctly speaking, the sources, of the acetone bodies as undecided.
We know just as little concerning their place of formation.

The occurrence of acetone bodies in the blood has for a long time been
considered to have considerable significance concerning the course of
diabetes. It has been assumed that the presence of acids decreases the
alkalinity of the blood, and thus brings about severe disturbances. This
is, in general, not the case, because the organism can form ammonia which
by combining with acids keeps the reaction of the blood, and thereby that
of the tissues, within normal limits. The fact that the interchange of
oxygen is not materially altered during the elimination of considerable
amounts of these aldehyde bodies, speaks in favor of this a.ssumption.
Sometimes, however, there takes place a rapid formation of large
amounts of these acetone bodies, resulting in considerable injury to the
whole organism, namely in coma diabeticum. By this name is meant a
complex of symptoms which very often occurs, in case intercurrent disease
has not carried off the patient, at the period near the death of the diabetic.
The condition is characterized by progressive dyspnoea, somnolence, and
sinking of the body-temperature. In many cases the patient recovers,
the acetone bodies are again eliminated in the urine, and the organism has
time to combat the acid by the formation of ammonia. After some time
the symptoms reoccur with increased severity, until finally the patient
dies as a result of such an attack. The first explanation of this was in
attributing a specific poisonous action to the acetone bodies. This is,
however, as direct experiments with the separate substances has shown,
not the case." Obviously we have here a true instance of acid poisoning.
The blood and tissue react acid after a diabetic has died in coma. There
takes place, together with this change in the reaction of the blood, a diminu-
tion in the processes of oxidation. The/8-hydroxy-butyric acid is no longer
oxidized to acetoacetic acid, or at least only to a slight extent. Both of
these acids unite with the free alkali in the blood. This explains the fact
that the amount of carbonic acid in the blood sinks during the coma. The
fact that there is le.ss oxidation in the organism during this period is proved
by the fact that various products of the hydrolysis of albumin are now

' Deut. Arch. klin. Med. 76 (1903).

• F. T. Frerichs: Z. klin. Med. 6, 3 (1883). P. Albertoni: Arch, exper Path.
Pharm. 18, 218 (1884). Stadelmann: ibid. 17, 419 and 443 (1883).

100 LECTURE V.

found in the urine.' Tiie general symptoms during coma diabeticum, as
Walter ' showed, have great similarity with those observed during experi-
mental acid poisoning. He injected dilute hydrochloric acid into the
stomach of rabbits — animals which, in contrast to all other animals which
have been investigated, are not able to combat acid by the formation of
ammonia — and dyspnoea soon appeared. The carbonic acid content of
the blood was greatly lessened, and the ammonia in the urine considerably
increased. By subcutaneous introduction of bicarbonate of soda the
symptoms were relieved, and the animal revived.

The formation of these acetone bodies is not restricted to diabetes. It
takes place also under certain conditions in all the different kinds of
glucosuria. There is always some disturbance in the metabolism of carbo-
hydrates, but there is not necessarily a direct relation between the two
symptoms. It must not be forgotten that actually such a deep-seated
disturbance of metabolism as exists both in the mild and chronic forms
of glucohemia can never exist by itself, i.e., be limited in its effect to one
class of substances. Since the metabolism of the organism may be traced
back eventually to the metabolism of its cells, it is easy to see how much
the whole metabolism must suffer if one group of its most important
nourishing and building materials becomes afflicted. It is readily
comprehensible that in the course of time the metabolic disturbance
becomes general, and the metabolism of the fats and albumins suffers as
well. It is from this point of view that we must regard the patient suffer-
ing from a severe type of diabetes, in order to get some idea of the whole
scope of the disturbance. It is not alone the loss in energy which goes
on during the constant carrying away of large amounts of sugar, and
which indeed may be compensated to some extent by other food-stuffs,
that governs the whole disease and makes it so serious, but the general
derangement of the entire metabolism. The abnormal composition of
the blood and of the lymph gives rise to many secondary phenomena,
the resistance of the tissues and cells to infection is diminished (the num-
erous tissues furnish a favorite nutriment for certain forms of life — •
furunculi, colonies of aspergilli, etc.) and thus one trouble follows another,
eventually giving to the disease of diabetes its peculiar characteristics.

' Abderhalden: Z. physiol. Chem. 44, 17 (1905).
' Arch, exper. Path. Pharm. 7, 148 (1877).

LECTURE VI.

FATS — LECITHIN — CHOLESTEROL. .

Among the foods of the animal organism, the fats occupy a peculiarly
important place, due to their high calorific value. They also play an
important role in the plant, and especially in the animal kingdom, as
reserve material. The animal organism stores tremendous reserves of
vital energy in the form of fats.

The most important locations are the intermuscular connective tissue,
the fatty tissues of the abdominal cavity, and the subcutaneous connective
tissue. Small deposits occur in every organ and cell. These fat reserves
vary much in size, depending on nutritive conditions, so that no definite
statement can be made regarding the fat content of the individual organs.

In the vegetable kingdom the fats are also widely distributed, and act as
reserve stores, yet never to the same extent as in the animal organism.
Fats are deposited in dormant parts, as in seeds, in which we first find
the accumulations of carbohydrates, then fats; often they occur together.
Here the fat does not occur in the form in which we meet it in the animal
tissues. It is very finely disseminated throughout the protoplasm, only in
isolated cases occurring deposited as crj^stalline aggregates in the nutritive
cells. Supplies of fat have occasionalh^ been found in stalks embedded
in the soil; onions, tubers, and roots, and similiarly the shoots and branches
of bushes in winter as well as the leaves of evergreen have shown a reserve
supply of fat.

The fats are composed entirely of the elements carbon, hj-drogen, and
oxygen. The natural fats belong to the group of neutral fats. Free fatty
acids are found only in small quantities. The neutral fats are, in general,
to be considered ' as esters of glycerol and monobasic fatty acids; i.e., in
the triatomic alcohol, glycerol, the hydrogen atoms in all three hydroxyl
groups are substituted by fatty acid-radicals (R) :

CHoO . R

CHO .R

I
CHaO^.R^

Glycerol Fatty acid
residue radical

' Cf. Chevreul: Chem. Untersuchungen iiber Tierische Fette. Paris, 1823.

101

102 LECTURE VI.

The three fatty acids, oleic, C18H34O2, stearic, C18H36O2, and palmitic,
C10H32O2, play the principal role in the formation of fats. The last two
belong to the series of normal saturated fatty acids, CnH2n02, whose lower
members are represented Vjy formic, acetic, and propionic acids. Oleic acid,
however, is an unsaturated fatty acid; CgHiy.HC : CH (CH2)7.COOH.
These fatty acids combine with the triatomic alcohol, glycerol, (com-
monly called glycerin) splitting off water. We speak of tripalmitin, tris-
tearin, and triolein according to the nature of the fatty acid concerned:

CH2OH C15H31COOH CH, . O . CO . C15H31

I I

CH . OH + C15H31COOH = CH . 0 . CO . C1SH31 + 3 H2O

I I

CH2OH C15H31COOH CH2 . O . CO . C15H31
Glycerol Palmitic acid Tripalmitin

Tripalmitin and tristearin are solid at the ordinary temperature, while
triolein, on the other hand, is liquid. As fats are mainly mixtures of the
triglycerides, the solid or liquid condition of these depends entirely on the
quantity of triolein present. Besides these separate triglycerides there
are also mixtures, for instance, dipalmito-olein, a fat whose glycerol base
is united to two molecules of palmitic and one molecule of oleic acid;
again, we have distearopahnitin. Aside from the fatty acids mentioned,
there are the volatile members of the normal series; i.e., butyric, caproic
(hexoic), caprylic {octoic), and capric (decoic) acids. Thus in vegetable fats
almost all the different members of the normal fatty acid series have been
found. Even the higher fatty acids, such as lauric (C12H24O2), myristic
(C14H28O2), and arachidic (C20H40O2), as well as individual oxy-fatty
acids, have been observed. The latter have been isolated with certainty
only in the vegetable kingdom, while in the animal body the fatty acids
may combine with the higher alcohols instead of with glycerin. Rohmann'
has shown that in the secretions from the oil-bags of birds a portion of
the fatty acids is combined with octadecyl alcohol, CH3 . (CH2) le • CH2OH.
The palmitic acid ester of cetyl alcohol, C16H33OH, from spermaceti, or
cetin, has long been known to occur in the cranium of the sperm whale.
The palmitic acid ester of myricyl alcohol (CsoHeiOH) is found in bees-
wax. Analogous combinations are widely distributed in nature. In only
a few cases, however, has their identification been positive.

While triolein and the glycerides of the lower fatty acids constitute the
major part of the vegetable fats, tripalmitin and tristeariil predominate in
animal fat. An exception occurs only in the fat supply of cold-blooded
animals, which is very rich in triolein, as a consequence of which it remains
in a fluid state at temperatures which would cause solidification among

■ Hofmeister's Beitriige, 5, 110 (1904).

FATS — LECITHIN — CHOLESTEROL. 103

warm-blooded animals. Among the latter there are many differences,
depending on the species. For instance, human fat melts at about 25° C,
mutton-tallow at about 50° C, and fat of the horse at about 65° C.

The fats decompose according to their composition, by taking on water,
into fatty acids and glycerol. This hydrolysis can be accomplished by
various agents, for instance by boiling with dilute mineral acids, or with
alkalies especially in the presence of alcohol, and, above all, by the action
of specific and widely distributed plant and animal ferments known in
this case as lipa-ses. The process of decomposing fats is called saponifica-
tion.^ If this be accomplished by the action of free bases, we do not
obtain free fatty acids, but their salts. These are called soaps. Although
the sodium aad potassium soaps are easily soluble in water, the fatty acid
salts of the alkaline earths (calcium, barium, and magnesium soaps) are
insoluble. The neutral fats are perfectly insoluble in water. If we shake
them long and vigorously with water, we obtain an emulsion, which, how-
ever, soon disappears, while the fat separates again on the surface of the
water. Absolutely neutral fats, i.e., fats which do not contain a trace of
free fatty acid, cannot be emulsified even by shaking with dilute, alkaline-
carbonate solutions. When any free fatty acids are present, however, an
extremely fine, permanent emulsion is obtained.

With few exceptions fats are of little influence in plant economy,^ being
of far less importance than the carbohydrates. As a whole, they form
reserve material. In seeds, like Helianthus and Curcubita, we notice the
stored-up fat quickly disappears on germination. Concurrently we observe
an appearance of free fatty acid. A splitting of fat occurs, as was first
shown by Sigmund,' by the action of a specific ferment, lipase, also called
steapsin. More recently, extensive investigations have been made on
the very active lipase occurring in the seed of the castor-oil plant.* This
ferment is active in a weakly acid solution. The saponification of the fat
evidently changes it into such a condition that it can penetrate into the
protoplasm, and through the cell-walls. Small amounts of fatty acid in
the presence of alkali suffice to produce an emulsion. It has been demon-
strated experimentally that these extremely minute globules are capable
of penetrating the cell-membranes, although it has not been decided
definitely how large a quantity of the neutral fat is saponified. Again,
nothing definite is known at present concerning the further behavior of
the fatty-acids, though Sachs has shown that their disappearance coin-

' J. Gad: Arch. Anat. Physiol. 1878, 179-187.

' Cf. F. Czapek: Biochemie d. Pflanzen, vol. i, pp. 115-126. Jona, 190.5.

' W. Sigmund: Sitzungsber. d. Wiener Akad. 99, 407 (1890) ; 100, 328 (1891); 101,
549 (1892).

* W. Connstein, E. Hoyer, and H. Wartenberg: Ber. 36, 3988 (1902). Connetein:
Arch. Anat. Physiol. 1905.

104 LECTURE VL

cides with an increase in the amount of carbohydrates. We shall come
back to this point later on.

Let us now follow the course of the fat, which is received by the animal
organism in its food, as it passes through the alimentary canal and through
the paths of absorption. Fat passes the entrance to the alimentary tract
(i.e., the mouth) in a perfectly unaltered condition. Saliva has no action
upon it, and, although saponification l)egins in the stomach, the extent to
which this is accomplished is still much in dispute.' The digestion of fat
while in the stomach is of small moment, for the action of the ferment is
soon lessened, and eventually stops entirely, on account of the acid
reaction of the stomach juices. The lipase requires, moreover, for its
maximum efficiency that the fats shall be in an emulsified state, a con-
dition which is rarely fulfilled in the stomach. The action of the gastric
juices is, however, of indirect importance, because the fat of meat is set
free by the digestion of the connective tissue. For these reasons there
is no absorption worth mentioning of the fats while they remain in the
stomach. The real digestion of fat sets in when it reaches the intestine.
Here, first of all, it undergoes a purely physical change. By the action
of the free fatty acids, and on account of the presence of the alkali salts
in the intestinal and pancreatic juices and the bile, the fat is first of all
subdivided. The fatty acids, which are deposited everywhere between
the tiny particles of fat, react with the alkaline carbonates present. Soaps
result which now tear the tiny particles apart, making them still finer, a
process which is assisted by the carbon dioxide set free by the neutraliza-
tion. An emulsion is formed. The purpose of this proce.ss may be two-
fold. We can imagine that the epithelial cells of the intestine absorb
these fine globules directly in the same manner as is done by the plant
cells. Again, it is perfectly possible that the chief advantage of the
emulsion is to present an enormously large surface to the lipase, thus
facilitating its action.

We are absolutely certain that ingested fat is decomposed into fatty
acids and glycerol. The fatty acids unite as much as possible with the
alkali present, thus forming soaps. A question yet to be settled is the
extent of saponification; i.e., how much of the total fat in the food is
entirely decomposed. Although Pfliiger- assumes that the hydrolysis
must be complete, i.e., that only fatty acids and glycerol are available for
assimilation, other investigators believe that only a portion of the fat is

' Cash: Arch. Anat. Physiol. 1880, 323. Ogata: ibiJ. 1881, .515. Volhard: Miin-
chen. med. Wochschr. 5 and 6 (1900); Z. klin. Med. 42, 414; Verhandl. deut. Naturf.
Aerzte, 1901, II, 2d half p. 43. A. Zinsser: Hofraeister's Beitrage, 7, 31 (1905).
A. Fromme: ihiil. 7, 51 (1905).

2 Pfluger's Arch. 80, 111 (1900); ihiil. 81, 375 (1900); ibid. 82, 303 (1900); ibid.
85, 1 (1901); 85, 1 (1901); 89, 211 (1902).

FATS — LECITHIN — CHOLESTEROL. 105

saponified, while another portion, consisting of finely divided globules, is
absorbed in this state. In spite of numerous investigations a clear con-
ception of fat digestion is not yet at hand. Radijewski,' in investigations
with dogs, showed that soap can be assimilated, and converted into fats.
To show that a complete hydrolysis of fat occurs, the work of Connstein '
has been cited. He fed a dog with lanolin. Tiiis is not saponified by the
usual saponifying agents, although it forms an extremely fine emulsion
when rubbed up with water. 97.5 per cent of the lanolin administered
in this form was evacuated unchanged, in the fteces.

Many fats require complete decomposition to make them available for
assimilation. Such fats are the ones whose melting-points lie above the
body-temperature. I. Munk ' has shown that 90 per cent of mutton-
tallow, melting at 49°, was utilized by dogs. Spermaceti, melting at 53°,
was also assimilated. The rapidity and extent of digestion are certainly
influenced by the melting-points of the fats. The fats of lowest melting-
points are utilized soonest and in the largest quantities.

Microscopic investigations have been made in the attempt to estimate
the extent of the fat decomposition in the alimentary tract, by determin-
ing at what place in the epithelial cells and walls of the intestine the fat
is present as such. In the first place we must state that the walls of the
intestine are a large factor in the assimilation of fats. In it the fatty acids
and glycerol molecules are reunited so that large quantities of fatty acids
and glycerol are prevented from entering the body. I. Munk'' has shown
this very clearly. He fed dogs with pure fatty acids, and found an
increa.se in the neutral fat, but no free fatty acid in the lymph taken from
the thoracic duct.

That the synthesis takes place in the walls of the intestine is certain
from the investigations of Perewoznikotf .'' He fed fatty acid and glycerin
to a fasting dog, and obtained in the epithelial cells of the intestine the
same microscopic appearances as when he administered fats. Will ° and
C. A. Ewald ' even observed a fat formation from glycerol and fatty acids
in a dissected intestine. Microscopical studies of fat assimilation have
not given uniform results. Some observers have found the basal edges
of the intestinal epithelial entirely homogeneous and free from fat globules
during the absorption of the fats, and could indicate their presence only

' Virchow's Arch. 43, 208 (18G8).

' Connstein: .\reh. Anat. Physiol. 1899, :iO; Die mod. Wocho, No. l."), 1900.
» Virchow's Arch. 95, 407 (1884). Anischiiik: Z. I5iol. 26, 434 (1890). O. Frank,
Arch. Anat. Physiol. 1894, 308. F. Miillcr: Z. kliti. .M.-d. 12, 4.5 (1887).
* Virchow's Arch. 95, 4.31 (1884'); ibid. 123, 230 and 484 (1891).
' Zentr. med. Wis.sen.sch. 1874, 851.
' Pfliiger's Arch. 20, 2.5.5 (1879).
' Arch. Anat. Physiol. 95, 407 (1884).

106 LECTURE VI.

in the second third of the cells; other investigators' have noted globules
in the basal edge. The results do not enable us to arrive at any decision.
It has also been stated that an active ingestion of unchanged fat globules
takes place.^ The epithelial cells are supposed to send out protoplasmic
processes which surround and absorb the fat globules. The leucocytes
have also been credited with a direct activity in the assimilation of the
fats. They are believed to migrate in the intestinal lumens and saturate
themselves with fat. Finally, there exists the possibility that other
methods may exist for fat assimilation. Aside from the fat absorption by
the cells, there remains the possibility of intercellular absorption.

For the time being, we can only state with certainty that fat is emulsified
in the intestines, and that there is always a decomposition of fat into
glycerol and fatty acids. The only question is as regards the amount of
fat saponified. Undoubtedly the decomposed fat is recombined in the
walls of the intestine, so that only neutral fats are introduced into the
organism.

Many factors participate in the process of fat absorption. One of the
most important is the pancreas. It furnishes, on the one hand, the alka-
line fluid which is so necessary for the emulsification of the fats, and,
again, it supplies the fat-splitting ferment, which produces the saponifica-
tion. If the amounts of the pancreatic juice be diminished, either by
extirpation of the gland, or through ligating the ducts of the pancreas, an
appreciable reduction in fat absorption takes place. It is not entirely
abolished, for Abelmann ' has shown an absorption of 28-53 per cent of
milk-fat under these conditions. Sandmeyer has shown that, if dogs
whose pancreatic glands have been removed are fed with finely chopped
pancreas, the amount of fat absorbed can be increased.

It is still a question whether the absence of lipase, which results
on the removal of the pancreatic juice, is the cause of the diminished
fat assimilation. If it be true that a copious emulsification is sufficient to
cause a fat absorption, we must conclude that this can take place without
the pancreatic lipase being necessarily present, because fatty acids are
certainly set free in the stomach, and fats can also be decomposed by
bacterial action. The formation of an emulsion might, on the other hand,
tend to prevent the diminution in amount of the alkaline pancreatic juice.
If we saturate an emulsion of fat with an acid, we observe that the emulsion

' Cf. I. Munk: Zentr. Physiol. 14, 6/7, 121, 152, 409 (1900). Heidenhain: Pfluger'a
Arch. 43, Sup. 85 (1888). Kischensky: Zentr. allg. Path. u. path. Anat. Heft 1,
1902; Beitrage z. path. Anat. u. z. allg. Path. 32 (1902).

■ Cf. Zawarykin: Pfliiger's Arch. 31, 2.31 (188.3). Heidenhain: he. cit. L. V.
Thanhoffer: ibid. 8, 391 (1874). R. Wiedersheim : Festsch. 56 Ver-sam. deut. Natur-
fors. Aerzte, 1883.

' Abelmann: Inaug. Dis,s. Dorpat, 1890. Cf. W. Sandmeyer: Z. Biol. 31, 12 (1894).

FATS — LECITHIN — CHOLESTEROL. 107

gradually disappears. Large oil-drops are formed, which collect on the
surface of the liquid. It is very probable that the diminished fat absorp-
tion is largely dependent on the reduced alkalinity of the pancreatic juice,
on account of the acid contents of the stomach preventing the formation
of an emulsion in the duodenum.

Although Teichmann ' has shown that if the pancreatic duct of a dog
be Hgated the absorption of fat is not apprecialily affected, this does not
invalidate our assumption. The secretions in the small intestines of the
herbivora occur in greater alkalinity than is the case with the carnivora.
That milk-fat is even assimilated in the absence of pancreatic juice is
possibly explained b}' the following circumstances. If milk is coagulated
by means of rennet, and the coagulum then dissolved in pepsin-hydro-
chloric acid, we obtain a very stable, acid fat-emulsion. It is rather dif-
ficult to obtain a clear conception of the actual relations of fat assimilation,
so long as the conditions of fat digestion are so little understood. It is,
of course, possible that the stomach lipase continues to act in the duodenum
even when the alkaline pancreatic juice diminishes, and in this way a part
of the fat is decomposed. We are certainly not justified in concluding,
from the fact that fat assimilation proceeds in the absence of lipase, that
neutral fats are directly absorbed.

The bile especially is of great importance in the absorption of fats.
Formerly, a direct influence on the intestinal epithelial cells was assigned
to it. It was supposed to stimulate them to assimilation. The function
of the bile, however, has been shown by Pfliiger ^ to consist of the ability
to produce solutions of the fatty acids and soaps. Large quantities of
stearic and palmitic acids were dissolved by a mixture of bile and sodium
carbonate. The cholates of the bile dissolve even the magnesium and
lime soaps which are insoluble in water. That the bile exerts a consider-
able influence on the absorption of fat is shown by the following observa-
tions: Dastre' ligated the bile duct of a dog, and made a fistula between
the gall bladder and the middle of the small intestine. With a diet rich
in fat the lacteals showed a milky turbidity below this fistula. The
bile acting alone does not seem capable of causing a fat assimilation,
although it acts more in conjunction with the pancreatic juice. This can
be shown very beautifully by an experiment upon a rabbit. In this
animal the bile duct joins the small intestine about ten centimeters above
the pancreatic duct. Between the junctions of these two passages the
chyle vessels remain clear and transparent, even on a diet rich in fat.
It is only below where the pancreatic juice enters that we notice the turbid
milkv streams of fat-bearing chyle.

' Inaug. Diss. Breslau, 1891.

' E. Pfliiger: Pfliigcr's .Vrch. 88, 299, 4.31 (1902); 90, 1 (1902).

' A. Dastre; .A.rch. phys. norm, et path. V. 22, .'515 (1890).

108 LECTURE VI.

Let us follow the progress of the fat, that has been absorbed by the
walls of the intestine, and which has undoubtedly been re-formed to some
extent, in its further passage through the organism. If we examine the
viscera of a fasting or starving animal, we observe the chyle proceeding
in a transparent vessel from the intestine to the mesenteric lymphatics.
We obtain an entirely different appearance if we feed the animal a diet
rich in fat just prior to death. The lacteals are then plainly visible. They
have become milky and opaque. If we investigate their nature, we find
that they are permeated with fat, even if no fat, as such, but only fatty
acids have been administered. In the latter case the glycerin was
necessarily missing for a fat synthesis, which must therefore be provided
by the organism in some other manner. If we make a fistula at the
entrance of the thoracic duct into the vena anonyma of a dog, we can esti-
mate the amount of chyle which escapes in a given period of time. In
a mixed diet we do not observe any increase in the quantity of chyle.
Its appearance only changes when the food contains fat. Although
ordinarily transparent, it then becomes white and opaque. In this process
of digestion the fats behave differently from other food materials, all of
which are poured directly into the blood-stream, and thence conveyed to
the liver. The chyle itself retains the fat in the form of a finely
divided emulsion.

I. Munk and Rosenstein ' observed in a girl, who was afflicted with a
fistula of the thoracic duct, that over 60 per cent of the fat consumed
flowed out of the fistula in less than twelve hours. Only about one
twenty-fifth of the fat administered had been saponified. Certainly, all
fats do not follow such an indirect course, for on feeding a diet rich in fat
a direct transmission into the blood-stream occurs. If the thoracic duct
is ligated, larger quantities of fat are carried into the blood. I. Munk and
Friedenthal- found that after a liberal consumption of cream, the fat con-
tent of the blood increased to six times the normal. As much as this had
passed into the blood, although only 32-48 per cent of the fat had been
assimilated. Fat also appeared in the blood after administering fatty
acids, about four-fifths of these having been converted into normal fat.

The amount of fat absorbed depends, as previously indicated, on its
composition. For instance, 97.7 per cent of olive oil is utilized, and
97.5 per cent of fats, whith melt at temperatures between 25-34 degrees
(goose-grease and lard). On the other hand, 90-91.5 per cent of mut-
ton-tallow, melting at 49-51 degrees, and only about 15 per cent of sper-
maceti melting at 53 degrees, are absorbed by human beings.

Pettenkofer and Voit ' as well as Rubner * have studied the absorption

' Virchow's Arch. 123, 230 and 484 (1891); Arch. Anat. Physiol. 1890, 37G and 581.

' Zent. Physiol. 16, 297 (1901).

» Z. Biol. 9, 1 (1873). * lb!J. 15, 115 (1879).

FATS — LECITHIN — CHOLESTEROL. 1 09

of fats. The former found that a dog, weighing 35 kilograms, assimilated
93 per cent of 350 grams of fat administered in a da}'. Rubner main-
tains that the intestine of a human being can absorb a like quantity. As
a rule, however, 100-120 grams is about as much as the system can
stand.

The blood also shows changes after the absorption of fat. Chyle, charged
with fat, is continually poured into the blood-stream through the thoracic
duct. The blood, and especially its plasma, quickly shows an increased
fat-content. This is especially true if the fat absorbed is large in amount,
and is indicated by a milky turbidity, of an otherwise clear plasma.
Often a distinct separation of drops of fat on the surface can be obtained
by placing such plasma in the centrifugal. Ultimately the excess of fat
again disappears from the blood. The process by which this is accom-
plished has not yet been demonstrated. The globules of fat do not
migrate through the capillary walls. It is possible that the leucocytes
have some function here. .\t one time it was considered certain that the
blood contained a lipase. Why it should be present, however, was not
clear, because, through the syntheses in the intestine, the organism protects
itself against free fatty acids. Why, then, should the fat be decomposed
again in the blood? This hypothesis has been abandoned, but on the other
hanil the investigations of Connstein and Michaelis ' have shown that blood
possesses the ability to transform fats into unknown substances, which
are soluble in water and capable of dialysis. This process is dependent
upon the presence of oxygen, and seems to require the interaction of the
red corpuscles. A part of the absorbed fat is taken directly to the tissue-
cells and consumed.

The unused fat is stored away as such. This is evident from the
following experiments. Franz Hofmann ^ allowed a dog to fast until it
was devoid of fat. The beginning of this period can be ascertained by
estimating the amount of excreted nitrogen. The starving animal utilizes
its stores of glycogen and then of fat, keeping its albumin intact as long
as possible. If the fat supply is used up, a rapid decomposition of albumin
takes place. The nitrogen elimination increases immediately. This occurs
in from four to si.x weeks. Hofmann then fed the animal under inspection
with considerable bacon and little meat. The amounts of fat and albumin
given were accurately determined. It was found that in five days this
dog assimilated 1854 grams fat, and 254 grams albumin; and stored up
1353 grams fat. This proved that fat in food is utilized to increase the
fat supply in the body. Pettenkofer and Voit ' reached the same conclu-

• ' Sitzungsber. Akad. Wissensch. zu Berlin, 771, 1896; Pfliiger's .\rch. 65, 473
(1S'J7) ; 69, 76 (1897). Cf. Ergebnisse d. Physiologic, 3, 1, 194.

' F. Hofmann: Z. Biol. 8, 153 (1872).

' hoc. cit. Z. Biol. 9, 1 (1873).

110 LECTURE VI.

sion by another method. They determined the amounts of excreted
products from a dog, which had been given a hberal fat diet, with but
Uttle meat. They found that all the nitrogen appeared again in the excre-
tions, but that all the carbon did not do so. I. Munk ' could also obtain
storage of fat in a starved dog, by fats, or even fatty acids.

The proof that nutrient fat and stored-up fat possess direct inter-
relations, has also been obtained in still another manner. For fourteen
days I. Munk ' fed a dog, weighing 16 kilograms after nineteen days' fasting,
on fatty acids obtained from mutton-tallow. The weight of the animal,
which had been reduced 32 per cent during the previous fasting period,
then showed an increase of 17 per cent. On dissecting the animal, a very
large fat addition was noted. On " trying out " this, about 1100 grams
of fat were obtained, which was solid at room temperature, and melted at
40° C. It is well known that mutton-tallow melts at this temperature,
while fat from a normal dog would possess a far lower melting-point
(about 20° C). I. Munk used rape-seed oil in a second experiment. A fat
was obtained which melted at 23° C. while at 14° C. a granular crystalline
deposit separated. The fat obtained showed 82 . 4 per cent oleic acid, and
12.3 per cent fatty acids. Normal dog-fat contains only 63.8 per cent
oleic acid, and 28 . 8 per cent solid acids. Rape-seed oil contains erucic
acid (C22H42O2). This was isolated from the above dog-fat.

It is very remarkable, that a food-stuff, and especially a vegetable one,
should determine the composition of an animal tissue. We shall see later,
that the decomposition of the organic food-stuffs not only makes it
possible for them to l^e absorlsed, but also enables the organism to select
the material necessary for its own development.^ In fact, the fatty tissues,
together with glycogen, maintain a distinct individuality when compared
with the other substances of the tissues. Both are reserve-materials
which the organism stores up, in order to utilize them when needed. We
do not, however, desire to place glycogen and fat in the same category.
Glycogen is of far more importance in metabolism, than is fat in the true
fatty tissues. It is continually being used up in metabolism, and also
being constantly redeposited. The fat supplies in the liver, and possibly in
other organs also, act similarly to glycogen. They likewise undergo quick
changes. The fatty -tissue proper, however, is a true tissue under ordinary
circumstances. Aside from its function as a reserve material it serves for
other purposes, e.g., a purely mechanical one (like the fat in the eye-socket,
etc.), and again as a non-conductor of heat. It must not be inferred that
the fat is deposited as a dead mass entirely protected from metabolism.

' Arch, f.- (Anat. u.) Physiol. 1883, 27.3; Virchow's Arch. 95, 407 (18S4). Cf. G.
Rosenfeld: Verb. d. 17, Kong. f. in. .\Icd. 503 (1899).

^ Cf. E. Abderhalden: Zentr. Stoffwechsel- Verdauungskrankheiten, 5, No. 24, 647
(1904).

FATS — LECITHIN — CHOLESTEROL. Ill

On the contrary, on leaving the blood-stream in a diffusible condition, at
present not understood, it is taken up by certain cells, and there converted
into fat. The fat-cells themselves are supplied with an efficient resisting
membrane. It withstands the action of alcohol and ether, and is not
dissolved by acetic and dilute mineral acids. The fat-cells also contain a
yellow color principle. Like all cells they contain protoplasm, for which,
however, there is only a limited space if the fat supply be large. The
fatty tissue is closely interwoven with that of the network of blood-
vessels, so that this valuable material may be quickly utilized when
necessary. We do not know how this reserve material is liquefied. It
has not yet been determined in what form the fat leaves the cell and enters
the blood-stream. Nervous influences undoubtedly control these large fat
supplies, so that the fat cells are in this way kept in unbroken relations
with the general metabolism. It is not improbable that the fat-containing
cells of the connective tissue (i.e., the protoplasm which they contain) play
in the metabolism of the fats, a role similar to that taken by the cells of
the liver in carbohydrate metabolism. As the latter build up glycogen
from grape-sugar and so protect the excess of carbohydrate from oxidation,
and in case of need either directly or indirectly bring about cleavage, so the
fat-cells withdraw from the blood the exce-ss of valuable fat material,
retaining it for a time, in order to set it free again for oxidation at the
required moment. The animal organism has an efficient supply of reserve-
material in the fat. It possesses double the calorific value of carbohy-
drates and of proteins. One gram of albumin gives 4 . 1 calories, one gram
carbohydrate gives Ukewise 4.1 calories, while one gram fat, on the other
hand, gives 9.3 calories. In fasting, the fat supply is very quickly drawn
upon. Ordinarily the normal organism keeps this supply very constant.
Gradually, equilibrium is established to a certain extent between the
amount of the food which is used as fuel and that used for the replace-
ment of tissue. This, of course, applies only to the mature organism.
In man, frequently, this equilibrium is disturbed. More fat is in many
cases being added constantly, so that the fat deposits grow far beyond the
physiological requirements, and finally a condition results which is vari-
ously known under the names of adiposity, obesity, and polysarchia. It
is impossible to distinguish sharply between such Jlcshincss and a physi-
ological reserve supply of fat. Only when difficult breathing and faulty
heart-action are indicated, does the condition become a pathological one.
The causes of obesity are unknown. Various conditions can lead to this
same result. There is unquestionably a physiological obesity, which arises
from a rich diet, and a palholoijical obesity, which occurs in spite of all
precautions. The latter form belongs to the class of metabolic derange-
ments, and can be traced to an abnormal metabolism of the cells. It is
certain, that, until we are better acquainted with the subject of physiological

112 LECTURE VI.

fat assimilation, we cannot get an exact explanation of the causes of
obesity. We are confronted by a condition which leads to many secondary
symptoms; above all we have to remember that a very large amount of
tissue has to be supplied with blood so that unusual strains are placed
upon the heart. The condition of those afflicted with obesity undoubtedly
depends upon their ability to satisfy these demands.

The functions of fats in the animal organism are not restricted to the
part that they play as direct or indirect nourishment. In the growing
individual the fat which is in the cells, and also otherwise distributed in
the true fatty tissues, and the substances which are closely related to it,
play a part the importance of which we cannot yet fully estimate. We
are acquainted with many materials which are absorbed in a water-soluble
condition, and in this form they penetrate the cells. On the other hand,
we also know of many substances which are entirely insoluble in water,
but which are, nevertheless, easily taken up. The fats may in these
instances act as solvents. Although a substance is soluble in water, it
may be more so in oils ; and this property may exert an influence on cellular
assimilation, and permit the cell to exercise a selection according to its
constitution and condition. This suggestion has been made by H.
Meyer ' and by Overton ^ to explain the action of certain narcotics. It is
possible, and in fact, even probable, that such relations are important for
cell-metabolism, under ordinary circumstances.

Closely related to the fats, and the functions which we have just con-
sidered, is the lecithin group. These, also, are combinations of glycerol
with fatty acids. Here only two hydroxyls are substituted by fatty acids
in the tri-atomic glycerol, while the third is replaced by a phosphoric acid
molecule which is also combined with the base, choline. The following
formula gives an idea of the constitution of lecithin, also called distearyl-
lecithin : ^

Glycerol radical

CH2— O— C18H35O

I
CH— 0— CgHssO

Fatty acid radical.

ICH2-O ^ ,

HO — PO Phosphoric acid radical.

I /C2H40^ '

Choline radical N= (CH3)3
^OH

' Arch. exp. Path. Pharra. 42, 109 (1899).

' Studien u. d. Narkose, etc., Jena, 1901. Cf. also H. J. Hamburger: Osmotischer
Druck u. lonenlehre in d. Medicin. Wiesbaden, J. F. Bergmann, 1904, vol. iii, 242.

' Cf. Diakonow: Zent. med. Wissensch. 1868, 4.38. F. Hundeshagen: J. pr. Med. 28,
219 (1883). E. Gilson: Z. physiol. Chem. 12, 585 (1888). A. Strecker: Ann. 148, 77
(1868).

FATS — LECITHIN — CHOLESTEROL. US

On saponification with alkalies, we obtain fatty acids, glycerol, phos-
phoric acid, and choline. Dilute acids have little action on lecithin.
The fatty-acid component varies. We are acquainted with lecithins con-
taining stearic, palmitic, and oleic acids. Even two different acids may
participate in the constitution. We have not yet succeeded in pre-
paring lecithin synthetically. As it is optically active, it must contain an
asymmetric carbon atom. We are justified in making certain deductions
regarding the method of grouping of the glycerol and combined radicals,
as indicated by R. Willstadter and Karl Liidecke.' The following formula
are possible ones:

CH2—O— Choline phosphate CH2—O— Fatty acid A

*CH —0— Fatty acid *CH —0— Choline phosphate

CH,—0— Fatty acid CH2—O— Fatty acid B

I II

Formula II onl}' contains an asymmetric carbon atom when the two fatty
acids are different. The investigators mentioned decided in favor of
formula I, because they succeeded in obtaining an optically active glycero-
phosphoric acid by hydrolysis. This is only possible when the molecule
has the following grouping: HO . CHg— CH . OH— CH2 . O . PO3H,.

*

The base choline is of much interest. It is a quaternary-ammonium
base, and has the following constitution:

/CH3
/CH3

^CHo— CHoOH
^OH

It is, therefore, to be considered as trimethylhydroxyethylammonium
hydroxide. Wurtz - proved this by synthesis. He combined ethvlene

/CH3
oxide, C2H4O. trimethvlamine N — CH3 and water. Choline can also be

^CH3
derived from glycol, as shown by the following formula:

CH^OH

I
CH2— N— (CH3)3

I
OH

■ R. Willstiitter and K. Liidecke: Ber. 37, 3753 (1904).

' Ann. Sup. 6, IIG and 197 (1808). Cf. M. Kriiger and P. Bergell: Ber. 36, 2901
(1903).

114 LECTURE VT.

In aqueous solution choline breaks down into glycol and trimethyl-
amine. It has also been found in a free state in plants. It is closely
related to another base, also found in plants, and especially in sugar-
beets, known as betaine, or oxyneurine. Its fornaula is :

COO.

I >MCH3)3

CHz^

It has been obtained from choline by oxidation. Other bases have been
isolated from various plants, which in part have been given characteristic
names; e.g., amanitine, from toad-stools; fagine, from buchu seeds, etc.
They are, however, all identical with choline. In toad-stools {Amanita
Muscaria), there is found besides choline, another base called muscarine,'
which is evidently an oxidation product of choline, and can also be obtained
from it by oxidation. It is commonly considered to be an 'aldehyde,
although its constitution has not yet been established positively:

CHO

I

CH2.N. (CH3)3

I

OH

Closely related to these is neurine, which has been isolated from the
brain by Liebreich.' Its composition is that of trimethylvinylammonium-
hydroxide :

xCHa

^CH = CH2
^OH

The second component of lecithin, the glycero-phosphoric acid, is easily
produced by uniting glycerol and phosphoric acid.

The lecithins are widely distributed in the plant and animal kingdoms.
We could truly say that every cell contains lecithin. It occurs particu-
larly in animal tissues, in the brain, nerves, fish-eggs, yolk of eggs, and
in spermatozoa. It is also found in the muscles and the blood (in the
plasma as well as in the blood corpuscles)^ in the lymph and leucocytes;
in fact, in every cell and in every organ. We find lecithin very widely

O. Schmiedeberg and E. Hamack: Arch. exp. Path. Pharmak. 6, 101 (1877).

Ann. 134, 29 (1865).

Cf. E. Abderhalden: Z. physiol. Chem. 25, 65 (1898).

FATS — LECITHIN — CHOLESTEROL. 115

distributed in the vegetable world, more especially in seeds. During
germination the lecithin content increases.'

In digestion, lecithin acts in an analogous manner to the fats; in fact, it
resembles these very closely in every respect. It forms an emulsion with
water. It partly resembles a colloid. Lecithin is decomposed by lipase
into glycero-phosphoric acid, free fatty acids and choline; it is not cer-
tain that the decomposition of lecithin in the alimentary tract is complete,
nor that unchanged lecithin can be directly absorbed. It is rather to be
assumed that its components are separately turned over to the organism
for further use.

The wide distribution of lecithin leads us to conclude justly that it is
of great importance to the animal organism. We, however, know little
about its function at present. From its constitution we can indeed
a.ssume that it acts as an intermediary body between various groups of
compounds. We easily recognize its relation to the fats, from which it
perhaps derives two components, the fatty acids and glycerol. On the other
hand, lecithin evidently acts as a bridge to the very important nucleins.
It is possible that lecithin plays a leading part in the internal metabolism
of the cells. To a certain extent it represents the fat of the cells.
Furthermore, it unites the inorganic foods with the organic ones. The
nucleins possiblj^ obtain their phosphoric acid from lecithin.

We do not know anything at present concerning the occurrence of
lecithin in the organs. It may be there in the free state, or it may enter
into numerous combinations. Many lecithides have been described, but
as lecithin has the property of readily enclosing other substances, e.g.,
albumin, all such claims should, for the moment, be regarded with con-
siderable skepticism.

The following experiments ^ may possibly give us some conception of the
functions of lecithin, even if only indirectly. If we remove every trace of
serum from the blood corpuscles by means of a centrifugal machine, and
careful washing with physiological sodium chloride solution, the corpuscles
are not dissolved by the cobra poison of the Naja snake, when suspended
in an isotonic sodium chloride solution. The process of dissolving the
blood corpuscles in such a way is called hemoli/sis, and the poisons
causing this are hemoliitic. If the serum is not separated from the
blood corpuscles they immediately go into solution on adding cobra poison;
i.e., the hemoglobin diffuses from the blood corpuscles into the surround-
ing medium. We can show the influence of serum in a better way
by taking thoroughly-washed blood corpuscles, suspending them in a

' Cf. E. Schuize and A. Lickiemik: Z. physiol. Chem. 16, 405 (1891).

' Cf. S. Flexner and H. Noguchi: J. Exp. Med. 6, No. 3 (1902). P. Kyes: Berl. klin.
Wochensch. .38 .39 (1902), Xos. 2-4 M903); Z. physiol. Chem. 41, 273 (1904). E.
Abderhaldcn and Le Count: Z. exp. Path. Therap. 2, 199 (1905).

116 LECTURE Vl.

sodium chloride solution, and adding only one drop of serum to this,
after having previously shown that cobra poison alone had not caused
hemolysis. S. Flexner and H. Noguchi, who first observed this fact, and
noticed it also with other poisons {tetanustoxin, solanin, saponin, etc.),
rightly concluded that some substance was undoubtedly present in serum,
which made it possible for the cobra poison to act on the hemoglobin of
the corpuscles. P. Kyes then succeeded in showing that lecithin could be
substituted in place of serum. Minute traces are sufficient to cause hemo-
lysis. Lecithin alone, when used in small quantities, does not act hemolyti-
cally, but lecithin and the cobra poison together do so. This is not the
place to dwell upon this interesting biological phenomenon and its expla-
nation. We must content ourselves with the knowledge that lecithin
possesses the capacity of accelerating the activity of poisons. Many
interesting questions are suggested by this fact. It is entirely possible
that lecithin also acts as an accelerator in the animal cells, and even on
the intracellular ferments. As a result of recent investigations we are
forced to conclude that the ferments as a whole are not released from the
cells in their active form, but that they require the influence of a second
substance to develop their activity. With such an hypothesis we can
easily explain the action of ferments in the cells.

To lecithin is ascribed a large influence in the construction of the cell-
walls, and also in the resorption of the cells. What was said concerning
the fat contents of cells is also applicable to this case. Lecithins act as
solvents.

There is another substance, which, although not at all related to lecithin
chemically, is like lecithin indispensable to all cells. This is cholesterol.
Its various modifications are widely distributed in the vegetable kingdom.
Vegetable cholesterins are designated phytosterols. In the animal organ-
ism it is found in all cells, in the blood, lymph, etc. It occurs in excep-
tionally large amounts in the brain and nerve tissues. In the gall bladder
it often gives rise to the formation of calculi, although this is almost
always a secondary effect, and a resultof disease of the bladder (catarrh, etc.)
It forms white, fatty-feeling crystals with a pearly luster. It is absolutely
insoluble in water. Sometimes cholesterol occurs in the free condition, as
in the blood corpuscles;' then again it forms ester combinations. For
instance, it is united with fatty acids - in the blood. Schulze isolated an
isomer of cholesterin from wool fat, called isocholesterol.^

The way cholesterol is formed is still unknown to us. We do not at
present know its constitution. All that we knew up to within a short

' E. Abderhalden: loc. cit.

' K. Hurthle; Z. physiol. Chem. 21, 331 (1895-96). E. Hepner: Pfliiger's Arch.
1898, 73.

' Ber. 5, 1075 (1872), and 6, 251 (1873).

FATS — LECITHIN — CHOLESTEROL. 117

time, is that the formula C27H44O, or C27H46O, may be assigned to it and
that the molecule contains a double bond and also an alcohol hydroxyl.
Recent investigations ' have shown that cholesterol belongs to a group of
chemical compounds widely distributed in the vegetable kingdom, but not
hitherto found in the animal economy. Cholesterol is evidently a terpene.
The animal organism, therefore, contains hydro-aromatic compounds.

At present, cholesterol is considered to occupy an isolated position in
the animal kingdom. In the vegetable world we could easily understand
its formation from the terpenes in a number of ways. From its consti-
tution, it hardly seems possible that cholesterol originates in the animal
organism. Animal cholesterol is undoubtedly vegetable cholesterol which
has been utilized by the animal organism for its requirements. We know
absolutely nothing about its decomposition in the animal body. Bond-
zyi'iski and Humnickni - have isolated a substance similiar to cholesterol
from human faeces, which does not possess a double bond, but has two atoms
of hydrogen more than cholesterol. About one gram of this compound is
excreted daily. It has been called " dihydro-cholesterol," or coprosterol.
The reduction is undoubtedly brought about by the activity of putre-
factive bacteria.

We know practicalh* nothing of the significance of cholesterol in the
animal organism. Its general occurrence leads us to conclude that it is of
great importance in cell metabolism. We cannot possibly consider it as
a decomposition product. We only know of one definite property of
cholesterol. This relates to the hemolytic action of lecithin and cobra
poison. We have seen that lecithin accelerates the activity of cobra
poison. Conversel}-, cholesterol retards the action of lecithin. We have
seen that if we add snake venom to blood corpuscles, suspended in watei;
and freed from serum, no hemolysis results; when, however, a trace of
lecithin is added, hemolysis quickly follows. If we then add a minute
quantit}^ of cholesterol suspended in methyl alcohol, the lecithin, which
previously had caused the cobra poison to become active, is now without
effect. The blood corpuscles are not dissolved. Lecithin and cholesterol
occur in all cells, and especially in the blood corpuscles. It is probable
that the}' also show their antagonism towards one another in these. We
are acquainted with various kinds of blood, whose corpuscles are dissolved
by cobra poison alone; others require the presence of lecithin. It is
perfectly possible, and even probable, that lecithin is present in these
different kinds of blood corpuscles in different states of combination,

' A. WindavLs: Ber. 36, .3752 (190.3); 37, 2027 (19041; 37, 3099 (190-1); 37, 475.3
(1904). O. Diets and E. Abderhalden: ibid. 36, 3177 (1903) ; 37, 3092 (1904). Cf. also G.
Stein: Inaiig. Diss. Freiburg, 1905.

' St. Bondzynski and V. Humnicki: Z. physiol. Chem. 22, 396 (1896-97); Ber. 29,
476 (1896). MiiUer: Z. physiol. Chem. 29, 129 (1900).

118 LECTURE VI.

or that cholesterol is present in a different form, or perhaps to a different
extent in one case than in the other. Perhaps when the cholesterol is in a
combined state lecithin may act normally; and conversely, lecithin may
be in some such state of combination that it is less active, so that in the
different processes of the cell at one time lecithin acts freely, while at
another it does not.

The terpenes, and especially the cyclic terpenes, are very widely dis-
tributed in the vegetable kingdom. Plant secretions are largely composed
of these. We are acquainted with a large number of the members of this
class. Limonene and pinene are most widely distributed. At present we
cannot say anything ' regarding their functions or their origin.

' Cf. F. Czapek: Biochemie der Pflanzen, G. Fischer, Jena, 1905, vol. ii, p. 658.

LECTURE VII.

ALBUMINS OR PROTEINS.

Elementary Composition. Simple Substances or Mixtures.
Classification.

The albumins, or proteins, occupy a distinct position among our organic
foods. They are indispensable, and cannot be replaced by either the
carbohydrates or the fats. They are large factors in cell-formation, and
possess just as important relations to the animal organism as do the carbo-
hydrates to the plants. We shall see later that the animal organism
obtains all its albumin requirements from the vegetables. With the
herbivora this requirement is supplied directh'; with the carnivora, indi-
rectly.

The albumins present a well characterized group of compounds. They
differ essentially from the carbohydrates and the fats in their elementary
composition. Besides the elements C, H, and 0, they invariably contain
nitrogen, and, as far as our present knowledge is concerned, also sulphur.
These five elements are found in proteins ' in closely agreeing amounts.
The carbon varies from 50-55 per cent, hydrogen from 6.5-7.3 per cent,
nitrogen from 15-17.6 per cent, oxygen from 19-24 per cent, and the
sulphur from 0 . 3-2 . 4 per cent. These figures, of course, mean but little,
and give us no conception of the composition of the individual constituents
of proteins. This fact must be thoroughly appreciated, because unfortu-
nately manj' far-reaching conclusions regarding the constitution and
identity of albuminous bodies have been made on the basis of the ele-
mentary analysis.

Before we proceed to discuss the composition of the proteins, we are
confronted with the question: Are we justified in considering albumin
itself as a well-defined, chemical individual? We shall see later that we
are acquainted with a large number of different proteins, which vary
according to their mode of formation, and, in part, their place of occurrence.
They all possess the common characteristic of not being able to diffuse

' The term proteid has been used in English as the equivalent for albuminous
substances (German, Eiweisskorper), although Hammarsten, Neumeister, and other
European authors have designated ius proteids what may be called " compound proteids."
It has seemed best, however, to follow the German text and to designate the whole
group as that of the proteins. — Tkansl.\tors.

119

120 LECTURE VII.

through animal or vegetable membranes. They belong to that class of
bodies designated by Graham ' as " colloids." If the colloid be liquid,
we call it a sol; while if it be solid, we designate it as a gel. Liquid and
solid gelatin represent these two phases. If the colloid is distributed
throughout water, in appearance practically dissolved, we call it in the
hydrosol condition. We are acquainted with many such colloidal sub-
stances among the inorganic compounds. Silicic acid is a good example
of this. If a solution of sodium silicate is treated with an excess of
hydrochloric acid, the silicic acid set free, remains apparently in solution.
If this is then transferred to a dialyzer, the excess of hydrochloric acid and
the sodium chloride produced in the reaction diffuses into the liquid — in
this case distilled water — which is placed on the other side of the mem-
brane. The silicic acid, on the other hand, remains behind, in the form
of a tough, viscous mass, which can be coagulated by introducing a few
bubbles of carbon dioxide gas. " Albumin solutions " act in an analogous
manner. If we transfer blood serum, which occurs as a pale yellow,
clear liquid, to a dialyzer, a floculent precipitate quickly separates out.
This is the globin of the serum, which separates, the salt which had held
it in " solution," having been withdrawn.

A question widely discussed, is this : Is the colloid occurring in the sol
form to be considered as an actual solution, or as a suspension? It is
variously answered. As the albumin solutions conduct the electric cur-
rent, the products in " solution " appear as both anions and cations, it
has been assumed that an actual solution exists.^ Nevertheless, there is
no sharp dividing line between a real and an apparent solution. We are
acquainted with all possible intermediate stages.'

As the colloids lose many of their characteristic properties by various
agencies, so, also, the albumins are easily deprived of their colloidal nature.
The process is called a " coagulation." It is irreversible. As we gener-
ally deal with the coagulated products in our investigations of the
albumins, we shall devote a little space to discussing the ordinary
methods employed in effecting coagulation. One of the most important
characteristics of albumin solutions is that of coagulating on heating.
One of the most important factors in the phenomenon of coagulation
is the amount of salt held in solution. We can heat an albumin
solution, which has been very carefully freed from salt by dialysis, and it
will not coagulate. If salt is then carefully added, albumin separates

' T. Graham: Philosophical Trans. 151, Part 1, 183, (1861).

' J. Sjoqvist: Skand. Arch. Physiol. 5, 277 (1895). S. Bugarsky and L. Liebermann:
Pfliiger's Arch. 72, 51 (1898).

' We have suspensions, colloidal solutions, and true solutions. It is easy to dis-
tinguish between the end members of the series, but no sharp distinction is drawn
between these three classes. — Th.^nslatgrs.

ALBUMINS OR PROTEINS. 121

out. The reaction of the hquid is of the greatest importance. Complete
precipitation of the albumin can only occur when the solution is jaintly
acid. Any e.xcess of acid will hold some albumin in solution. Yet a
coagulation will take place on heating. The coagulated albumin has
combined with the acid. A so-called " acid-albumin " results. The
presence of alkali will prevent the coagulation of albumin for the same
reason. "Alkali-albuminates " are formed. The coagulated albumins are
insoluble in water and in neutral salt solutions. The readily soluble com-
binations of albumins with alkali and acids can be precipitated by salts.

The coagulating temperature of the different albumins varies. Efforts
have often been made to utilize this fact in separating the various albu-
mins. The method is inefficient. For one thing the albumins in solution
have different effects upon one another, and then, again, the coagulating
temperature varies considerably with the composition of the solvent.

We are also acquainted with other methods of effecting coagulation
besides that of heating. A number of albuminous bodies, for instance,
globin, myosin, antl fibrinogen, will go over into the gel form simply on
standing. Many precipitants, like alcohol, acetone, metallic-salt solutions,
etc., will also produce the same result. The time required to effect coag-
ulation varies considerablj-. For instance, the albumins are not immedi-
ately changed to their insoluble forms, on being salted-out. They can be
filtered, put into solution again, and in this way purified, provided that
these processes are carried out in a short time. .'According to Ramsden,'
coagulation has even been accomplished simply by shaking.

The most varied albuminous bodies assume very analogous physical,
and even chemical, properties after being coagidated. They all form an
amorphous powder, which is insoluble in water and salt solutions. We can
easily appreciate the fact that such material when used as a starting-point
for our investigations of the albumins gives little guarantee of purity or
homogeneity. We know that the colloids possess the property of carrying
down other substances from solution when they themselves are precipitated.
This is also the case with the albumins. They also contain appreciable
amounts of ash. It is not yet certain whether ash is an essential constitu-
ent of albumin or not; we do know that the amount can be decidedly
diminished by dialysis or by other methods.

As a rule, when we are investigating the composition of a new sub-
stance, and finally its constitution, the utmost precautions are used
to insure purity. To do this we usually resort to crystallization. By
re-crystallizing, and fractional crystallization, adhering substances are
removed. This accomplished, we analyse the substance in order to
determine its composition. The size of the molecule is obtained by a
molecular weight determination; and then by preparing a series of deriva-

' Arch. Anat. Physiol. 1894, 317.

122 LECTURE VII.

tives, decomposing the substance, and by other means, we arrive at the
constitution of the body in question. We consider that the constitution
of a substance is definitely settled only when, by synthesis, we succeed
in reproducing the same substance. We must attempt to follow this
same line of procedure in our investigations with the albumins. We now
turn to the question of crystallizing the albuminous bodies. Crystals of
albumins have been known for a long time. T. Hartig, in 1850,'
noticed crystalline substances in gluten meal, which are called aleuron grains
"protein granules," or "plant crystalloids." The albuminous nature of
these crystals was established by Radlkofer.- They have been observed
in many seeds; for instance, in pumpkin seeds, hemp seeds, ricinus
seeds, and especially in the Brazil nut. A beautiful example of this
kind of crystallization is presented by parasitical plants of the order
Orobanchacew, the tooth-wort,' Lathrcea squamaria. The cell kernels con-
tain protein crystals. A vigorous discussion has arisen as to whether these
substances are real crystals, or whether they only possess a crystalline
appearance. They possess characteristics which do not correspond with
those of true crystals. In the first place, these crystalline-appearing
substances swell up under the influence of water, and also of dilute
alkali. The refractive index of the crystal then diminishes. The
crystalline form also changes, because it does not expand uniformly
along its various axes. They are also partially soluble in glycerin.
A solid homogeneous residue remains, which retains the form of the original
crystal. There has been much discussion about this phenomenon. Fr.
N. Schulz * has indicated an interesting analogous example of an inorganic
crystalline formation. If human urine is allowed to stand for 24-48
hours with dicalcium phosphate, and then filtered, a precipitate of crys-
tals, one-half mm. in size, appears when the liquid is allowed to evap-
orate of itself. The crystals are like honestone with ragged points, and
they are strongly refractive (in polarized light, doubly refractive). If
these crystals are treated with dilute acetic acid a part is dissolved.
A crystal remains, however, which has the form of the original. It is
now singly refractive towards polarized light, and has lost its former
high refractive index. The dissolved portion is calcium phosphate; the
remainder, calcium sulphate. It is possible that the protein crystals
mentioned possess analogous characteristics. There is, however, nothing
further known at present to warrant these substances being classed

' T. Hartig: Bot. Zeit. 50, 881 (1850).

' L. Radlkofer: Ueber Kristalle proteinartiger Korper pflanzlichen iind tierischen
Ursprungs, W. Engelmann, Leipzig, 1859.

' A. F. W. Schimper: Diss. Strassburg, 1878; Z. Kristal, 1880. F. N. Schulz: Die
Kristallisation von Eiweissstoffen u. ihre. Bedeutung f. d. Eiweisschemie, G. Fischer,
Jena, 1901.

* Fr. N. Schulz: loc. cit, p. 4.

ALBUMINS OR PROTEINS. 123

with normal crystals. Such products have also been observed in animal
tissues. Thus, six-sided plates have been noticed in the intestinal
epithelium of the meal-worm, Tenebrio molitor} R. List ' states that
he has observed rhombohedrons and hexahedrons in the pigment cells of
the radial nerves of Spharechinus granularas, which gave the albumin
reactions. The small yolk plates, as well as other rectangular and quad-
rangular plates obtained from the eggs of fishes and amphibora, also be-
long to this class. Such products have also been observed in the eggs of
the roe' as well as in the epithelium of the tastes in man.*

These discoveries do not lead to anj' decision regarding the crystalline
qualities of the albumins. The fact that some of the products observed
gave albumin reactions does not prove that they were albumins. Small
impurities of albumin might have caused them. We would be but little
benefited even if the fact should be established that these crystalline
substances were albumins. The only value of crystals lies in possibility
of recrystallization and purification.

As a matter of fact it has been found possible not only to obtain many
of the albumins in crystalline form but many of them have also been re-
crystallized. Maschke ^ was the first to interest himself in this direction. By
evaporating a saturated solution of Bertholletia (Brazil nuts), he ob-
tained aleuron crystals in six-sided, tabular prisms.

Schmiedeberg " continued this work. The protein crystals were isolated
from the Brazil nuts by washing them out with petroleum ether. They
were then dissolved in distilled water at 30-35 degrees, and precipitated
by passing carbon dioxide into the solution. The precipitate was re-
dissolved by treating it with an excess of magnesium oxide at 30-35
degrees. By careful concentrating the solution, small crystals, of the
size of poppy seeds, settled out. These contained 1.4 per cent MgO.
Drechsel ^ improved this method considerably. Instead of evaporating he
removed the water by dialysis with absolute alcohol.

After grinding a large number of seeds and removing the fat, octahe-
dral crystals have been obtained by means of a five per cent salt solution
at 60 degrees. They can be redissolved and again precipitated. Such
crystals were obtained from cotton seed, hemp seed, and sun-flower
seeds.

' J. Frentzel: Arch. mik. Anat. 26, 287; Bed. entomol. Zeit. 26, 18S2. W. Bieder-
mann: Pfliiger's Arch. 72, 105 (1898).
' R. List: Anat. .\nzeiger, 7, 18.5 (1897).

' V. V. Ebner: Sitzung.sber. Akad. Wissensch. zu Wien. 110, part 3 (1901).
' Lubarsch: Virchow's Arch. 145, .317 and 362 (1896).
' O. Maschke: J. prakt. Chem. 74, 4.30 (18.58).
• O. Schmiedeberg: Z. physiol. Chem. 1, 20.5 (1877).
' E. Drechsel: J. prakt. Chem. 19, 331 (1879).

124 LECTURE VII.

It is a matter of great importance that albuminous bodies have been
crystallized which do not exist in the crystalline form in nature.
Hofmeister ' succeeded in crystallizing egg-albumin by treating a given
volume of egg-white with an equal volume of a cold, saturated solution of
ammonium sulphate. The precipitate consisted of globulins, while the
solution contained the albumins. By concentrating the filtrate from the
globulins at the usual temperature, beautiful microscopic needles were
obtained, which could be redissolved in a dilute ammonium sulphate
solution, and obtained again by evaporation. The precipitation of these
crystals can be greatly accelerated by making the solution faintly acid,
by adding either dilute acetic, sulphuric, or hydrochloric acids.^ Serum
albumin has been crystallized in the same manner.' Albumin from
horse-blood serum has also been obtained in crystalline form. We will
mention the fact here, that other albuminous substances are said to
have been crystallized, such as casein, lactalbumin, etc. We do not
need to dwell longer on this subject, for the investigations are not very
convincing.

We are acquainted with a protein which has itself not yet been oljtained
in crystalline form, although one of its compounds which occurs in nature
can be crystallized easily. We refer to the coloring matter of the blood,
which is a compound of the albumin globin, and another substance,
hematin, which is not of an albuminous nature. Hiinefeld * noticed a
crystalline separation when blood was dried between two glass plates.
Reichert, ^ however, is credited with being the true discoverer of oxy-
hemoglobin crystals. He observed them on the placenta of a nearly
mature guinea-pig foetus and also on the mucous membrane of the uterus
of the mother. Oxyhemoglobin may be prepared in various ways. The
most satisfactory method consists in centrifugalizing defibrinated horse-
blood, pouring off the serum, and washing the paste of blood corpus-
cles with isotonic salt solution until perfectly freed from serum. The
blood corpuscles are mixed with 2-3 times their volume of water at
30-35 degrees and the solution strained. In order to remove the stro-
mata of blood corpuscles, the solution is cooled to 0 degrees, shaken with
ether and one-quarter of the total volume of absolute alcohol, also at

' F. Hofmeister: Z. physiol. Chem. 14, 165 (1889); 16, 187 (1891).

' F. G. Hopkins and S. N. Pinlsus: J. Physiol. 23, 130 (1898). H. T. Krieger:
Diss. Strassburg, 1899.

' A. Giirber: Sitzungsber. physikal-med. Gesellsch. zu Wiirzburg, 1894, 143. A.
Michel: Verhandl. d. physikal-med. Gesel. zu Wurzburg, 29,28, No. 3 (1895), and Diss,
Wiirzburg, 1895,

* F. L. Hunefeld: Der Chemismus in der tierischen Oxydation, F. A. Brockhaus,
Leipzig, 1840.

s B. Reichert: Arch. Anat. Physiol. 1849, p. 197; 1852, p. 71. Cf. Fr. N. Schulz:
loc. oil. p. 23.

ALBUMINS OR PROTEINS. 125

0 degrees.' The crystalline separation of oxyhemoglobin suddenly occurs
after standing for some time on ice. As the solubility of the oxyhemo-
globins varies according to their animal origin, it is necessary to use
varying quantities of water for dissolving them. Thus, in order to
obtain the oxyhemoglobin of the cat, it has been found convenient to
use an equal volume of water in dissolving the blood corpuscles.'
Crystals may also be obtained by salting out with ammonium sulphate,
and dialysis with alcohol. Hemoglobin ' and methemoglobin * can also
be obtained in crystalline form.

Oxyhemoglobin can be redissolved in water at 37-40 degrees, and
recry.stallized by the addition of alcohol in the manner previously described.
When obtained from different animals it crystallizes in various forms:
thus the crystals obtained from the squirrel are hexagonal; those from the
horse are orthoi-hombic.

Crystals from insect blood have also been described. H. Landois *
has obtained crystals from the blood of caterpillars, Pupidoc, beetles, and
wasps, simply by evaporation. It is questionable whether these were albu-
min crystals. Crystals have also been isolated from the red sea-algae,
Rhodophijcew, or Floridecc. The Cijanophi/ceo' give a crystalline coloring
matter called phycocyan. More exact knowledge regarding the compo-
nents of these albumins is not yet at hand.°

Not only are all the external appearances of these crystals identical with
those of real crystals, but crystallographical investigations have shown
no differences. With the exception of those albuminous " plant crystals "
which belong, in part, to the regular system, they are all doubly refractive
towards polarized light. As a whole, however, only a few exact optical
investigations of albumin crystals have been made.

We have intentionally devoted considerable space to the discussion of
these individual crystals. It is a matter of the greatest importance to us
in our inve.stigations into the chemistry of albumins. Are we justified,
or not, in characterizing a protein substance, as pure? Let us see what
conclusions we can draw from the crystallizability of the proteins. The
attempt has been made to decide whether a protein was homogeneous by
means of an elmentary analysis. We have already shown that it is out
of the question to decide the question in this way. It is a well-known

' Cf. F. Hoppe-Seyler: Med.-chem. Untersuch. Vol. 2, p. 181, 1SG7. O. Zinoffsky: Z.
physiol. C'hem. 10, IG (1885).

' E. Abderhalden: Z. physiol. Chem, 24, 545 (1898). Cf. also Fr. Kruger: Z. Biol.
26, 409 (1890), and Z. physiol. Chem. 25, 256 (1898).

' Cf. G. Hiifner: ibut. 4, .{82 (1880).

' G. Hiifncr and J. Otto: ibid. 7, 65 (1882); 8, 366 (1884). A. Jaderholm: Z.
Biol. 20, 419 (1884).

' Z. wiss. Zool. 14, 55 (1804).

• Cf. H. Molisch: Bot. Zeit. 1894, 177; 1895, Til.

126 LECTURE VII.

fact that the most varied albuminous substances have very similar elemen-
tary compositions. It is impossible to recognize the presence of any
foreign albumin in a mixture by any such procedure. There is also the
added objection, that the same mixture will invariably be obtained by
following out a prescribed method.

It is very instructive in this direction that oxyhemoglobin crystals show-
ing no indication of any admixture under the microscope, may, neverthe-
less, be impregnated with foreign protein.' This fact has been established
through the discovery that glycocoU was present in a globulin from serum,
whereas oxyhemoglobin does not show the least trace of this amino-
acid. It has also been shown that glycocoll appears in the oxyhemoglobin
of horse-blood, after one crystallization. Another recrystallization gives
us a preparation entirely free from glycocoll. In this connection we would
refer to the various descriptions of albumins containing carbohydrates,
even when the observations were made with crystalline preparations.^

The crystallization of albumins does not correspond with the ordinary
formation of crystals from other sources. Most of the albumin crystals
are obtained by withdrawing the solvent. The production of these
crystals does not gradually follow the removal of the solvent. The crys-
tallization is, on the contrary, a very sudden one. This can be best
illustrated by salting out with ammonium sulphate. A very slight excess
is sufficient to throw out large quantities of crystals from an otherwise
clear solution. It is certainly a matter of great importance that no albu-
min as such has yet definitely been isolated in a crystalline state. A
possible exception would be that of the globulins, generally called edes-
tin, separated from plant seeds. They contain a considerable amount
of sodium chloride. It is impossible to remove this entirely, and still
retain the crystalline form. Although we are still in the dark, regarding
the influence of sodium chloride on the crystallization of edestin we do
know that the egg-albumin and serum-albumin do not themselves crystal-
lize, whereas their sulphates do, as was shown l:iy K. A. H. Morner.^ The
crystallization of the globin in hemoglobin depends on the presence of
hematin.

When we remember the extreme difficulty experienced in obtaining
absolutely pure crystals of substances of even low molecular weight, we
can hardly expect to obtain i-eally pure products through any methods
which in themselves can give no guarantee of efficiency. Although we
thoroughly appreciate the advantage of possessing the albuminous body
in a crystalline condition, we likewise find it necessary to state that not
the least confidence can be placed in the method of obtaining the crystals,

■ E. Abderhalden: Z. physiol. Chem. 37, 484 (190.3).

' E. Abderhalden, P. Bergell and T. Dorpinghans: Z. physiol. Chem. 41, 530 (1904).

' K. A. H. Morner: Z. physiol. Chem. 34, 207 (1901).

ALBUMINS OR PROTEINS.

127

nor even the system of crystallization itself, as a guarantee of the purity
of the individual substance. The only advantage that ciystallization
possesses is that it gives us a means of separating one product from the
mixture, and possibly increasing its purity.

The fact that we have so far not succeeded in obtaining a single abso-
lutely pure, individual, albuminous body, places the whole subject of
albumin investigation in a very uncertain light. At every turn we meet
this same unfortunate condition. We emphasize this point because a very
large number of investigations in the domain of albumin chemistry have
but little value for this reason.

This is especially true of the molecular weight determinations of albu-
min.' These have been carried out in various ways. The elementary
composition has been investigated to see if this would establish anything,
considerable attention having been given to the sulphur content. If
albumin considered as " pure," contains one per cent of sulphur, then
the molecular weight of the substance must be at least 3200 times that of
hydrogen. This method gives us only the minimum value, as we have no
means of knowing that only one atom of sulphur is present in the molecule.
The amount of sulphur in the various albumins differs greatly. The
following calculations have been made: "

Sulphur in Per
cent.

Molecular Weight: (with
the assumption that
each albumin mole-
cule contains one atom
of sulphur).

Edestin (cry-stallized)

Oxyheinoglobiii (liorse)

Seruni-albumiii (crystallized, horse)
Egg-albumin (crystallized) ....
Globulin

3680
7440
1700
2460
2320

Taking into consideration the fact that the albumin may contain more
than one atom of sulphur, Fr. N. Schulz ^ has estimated the molecular
weight of serum-albumin to be 5100, egg-albumin 4900, oxyhemoglobin
14.S00, globulin 4,600, edestin 7,300.

The substituted albumins, especially hemoglobin, give us another method
for estimating the molecular weight. Oxyhemoglobin contains, besides
the albumin globin, a substance containing iron, called hematin. About
0.4-0.5 per cent iron is present in oxA'hemoglobin. Every hematin mole-
cule contains one atom of iron. It is generally considered that oxy-

' Cf. Fr. N. Schulz: Die Grosse d. Eiweissmolekuls, G. Fischer, Jena, 1903.
' Fr. N. Schulz: loc. cil. p. 17.
' Fr. N. Schulz: loc. cit. p. 29.

128 LECTURE VII.

hemoglobin contains one hematin molecule and one molecule of globin,
an assumption for which adequate proof is lacking. A percentage of iron
of 0.4-0.5 per cent indicates a molecule of 14,000-11,200; a sulphur con-
tent of 0.43-0.67 per cent gives a molecule of 14,899-9500; and 4-5 per
cent of hematin ' one of 14,800-11,800.

Another method for estimating the molecular weight of proteins depends
on the formation of metallic compounds.

Harnack " has shown that many proteins can be precipitated from
solution by means of copper sulphate. We obtain a precipitate contain-
ing copper, called copper albuminate. Harnack obtained the following
amounts of copper in the precipitates from egg-albumin: (I) 1 . 34-1 . 37 per
cent Cu, and (II) 2 . 48-2 . 73 per cent Cu. Two different copper albumi-
nates were formed therefore. We are not acquainted with the conditions
governing the formation of one or the other compound. Copper albumin-
ate (I) would have a molecular weight of 4700, while the second compound
probably has the same value, if the assumption of Harnack is correct,
that both albuminates represent the same protein substance, differing
only in the fact that the first pos.sesses one atom, while the second has
two atoms of copper in the molecule.

Other metallic albuminates, such as those with silver, calcium, etc.,
have been prepared. It is difficult to state whether these are salt-like
compounds, or not. Recent investigations on the colloids have indicated
the necessity of being extremely cautious in passing judgment on such
compounds. Zsigmondy ^ has called attention to a peculiar property of
a colloidal gold solution, in the presence of albumin. A pure gold solution
is coagulated by an addition of electrolytes; for instance, sodium chloride.
If, however, albumin is present, the precipitation does not occur. The
albumin protects the colloidal gold. Fr. N. Sehulz and Zsigmondy * have
found it possible to express the degree with which each individual albu-
min protects the colloidal gold numerically. Thus globulin, under certain
conditions, can protect twenty times its weight of gold. If an albumin
solution, mixed with a gold solution, is precipitated, the gold is dragged
down. A homogeneous red precipitate is obtained. If the globulin be
redissolved, the gold will likewise go into solution. It can easily be seen
how such a behavior might lead one to believe that there is a compound
of albumin and gold, and thus to erroneous conclusions. It is of great
interest to know that crystallized egg-albumin also takes up gold, and
that the mixture can then be recrystallized. Copper, iron, calcium oxide,
etc., can also be held in colloidal solution by means of albumin.

" Fr. N. Sehulz: Z. physiol. Chem. 24, 449 (1898).

2 E. Harnack: Z. physiol. Chem. 6, 198 (1881).

3 R. Zsigmondy: Z, anal. Chem. 40, 597 (1901).
• Hofmeister's Beitrage, 3, 137 (1902).

ALBUMINS OR PROTEINS. 129

These statements are sufficient to show how little value should be
attached to the molecular weight determinations of such " compounds."
In individual cases we are not able to decide at present whether there is
an actual chemical combination, or whether the metal is merely held in
solution. No better results have been obtained by using halogen substitu-
tion products for the molecular weights. We are still without the neces-
sary knowledge and foundation for such work.

It might be thought that the cleavage-products could be utilized for
determining the molecular weights. Unfortunately, we have not j'et
sufficiently perfected our methods to utilize any individual, character-
istic, cleavage-product for such a determination. We must temporarily
content ourselves with approximations.

If we take all the known facts concerning the size of the albumin molecule
into consideration, and critically examine them, we must conclude that no
definite statement can be made. It is, of course, possible that the molecu-
lar weight is as large as has been computed; on the other hand, it may be
much smaller. It is, therefore, practically usele.ss to assign definite
formula to individual proteins as long as the methods of molecular weight
determinations are still so indirect, and based upon so many unknown
factors.

Direct determinations of the molecular weights of proteins have so
far been unsuccessful. The raising of the boiling-point method is not
applicable, because most of the albumins undergo changes on heating.
Determinations made up to the present by means of the lowering of the
freezing-point have not taken sufficiently into consideration the amount
of ash in the proteins. They are, therefore, practically worthless.

Before discussing the decomposition products of the albumins, or con-
sidering the question of the constitution of the albumins, we will now
devote a little attention to the various kinds of proteins as far as they can
be characterized by our present methods. It is impossible now to cla.ssify
the large number of known proteins in accordance with purely chemical
principles. We are still forced to follow the old grouping. This classi-
fication IS, however, only accepted as a matter of nece-ssity. The farther
we proceed into the chemistry of the albumins, the more we learn of
properties which can be utilized to identify individual proteins. We can
even indicate a prospective classification of the proteins according to their
constituents in an objective and satisfactory manner. We shall presently
see that thej^ are essentially composed of' amino acids. These are of very
different kinds. We distinguish between the mono-amino and the di-
amino acids. The relative amounts of these two groups of amino acids
vary considerably in the different proteins. We know of proteins like silk,
elastin, etc., which are mainly composed of mono-amino acids, the di-amino
acids being of little importance. On the other hand, we have the prot-

130 LECTURE VII.

amines, which are almost entirely built up of di-amino acids. There are
many intermediate stages between these two groups; thus, the histons
contain more di-amino acids than the above substances, which are rich
in mono-amino acids, although less than the protamines. The common
proteins, albumin, globulin, etc., occupy an intermediate position between
the silk, elastin, etc., group and the histons. In this way we may classify
the proteins as follows:

1. Proteins with less than 10 per cent of di-amino acids — elastin,
silk, etc.

2. Proteins with about 10 to 15 per cent of di-amino acids — serum-
albumin, serum-globulin, casein, etc.

3. Proteins with from, say, 20 to 30 per cent of di-amino acids — histon
from the thymus.

4. Proteins with larger amounts of di-amino acids (sometimes 80 per
cent or more) (protamines: salmine, clupein, etc.).

Our present knowledge is too inadequate for us to classify all proteins in
this way. The boundaries of the different groups are not sharply defined,
and we observe all sorts of intermediate stages between them. Further-
more, there is no doubt but that a member of one group may be trans-
formed while in the tissues so that it belongs to a different group. F.
Miescher ' has called our attention to an interesting example of such a
transformation. It is well known that, as the spawning season approaches,
the salmon journeys from the ocean into fresh-water streams. During the
entire period of several months in which it remains in the fresh water, the
fish eats nothing. On leaving the salt water it is a powerfully muscular
fish. These muscles are required for the stemming of strong currents.
Its sexual organs — testes and ovaries — are immaturely developed.
Gradually, however, the large lateral-dorsal muscle becomes smaller,
while the sexual organs assume large dimensions. There can be no
doubt but that the latter develop at the expense of muscular tissue. The
mature testis contains a protein rich in di-amino acids, known as salmine.
There is but little of this substance present in the immature organ. It
then consists chiefly of a histon-like substance. Histons, as a rule, do not
occur in any considerable amount in the muscles. It is very probable
that the protein in the muscles of the salmon loses di-amino acids, thus
increasing the proportion of di-amino acids in the protein, eventually
producing protamine with the intermediate formation of a histon. It is
highly interesting for the development of our knowledge concerning the
metabolism of proteins that we should study such relations further.

There is no question but that we shall shortly be able to classify the
proteins according to chemical principles. We must determine in the

' Die histochemischen und physiologischen Arbeiten von Friedrich Miescher.

ALBUMINS OR TROTEINS. 131

first place what amino acids take part in their formation, and then even-
tually stereochemical studies will decide the question. We have to
depend at present chiefly upon physical differences. Although many
proteins may be fairly well characterized in this way, by their solubility
in water, in salt solutions, etc., there are many others in which this is not
the case.

The proteins, as a whole, may be first of all separated into two main
groups: — (I) The Simple Proteins, and (2) The Compound Proteins, or
Proteids. The first group is subdivided into the true albumins and the
so-called albuminoids. The true proteins comprise (a) the albumins
(serum-albumin, egg-albumin, and lactalbumin, etc.); (&) the globulins
(serum-globulin, egg-globulin, lactoglobulin, and cell-globulin); (c) the
plant-globulins and plant-vitellins; (d) fibrinogen; (e) mj'osin; (/) phos-
phorized proteins, or the so-called nucleo-albumins (casein, vitellin, nucleo-
albumina of cell-protoplasm); (g) histons; {h) protamines. While these
groups are fairly well distinguishable, as we shall soon see, the following
group, which likewise belongs to that of the simple proteins, is charac-
terized more from a morphological point of view. The albuminoids
include (a) collagen; {b) ceratin (from hair, feathers, horn, etc.); (c)
elastin; (d) fibroin (from silk); (e) spongin and conchiolin; (/) amyloid;
(g) albumoid and perhaps the melanins.

To the compound proteins, or proteids, belong nucleoproteids, hemo-
globin, and glucoproteids.

In the following pages we shall merely devote space enough to such
description of the individual proteins as seems absolutely necessary for
later discussion. Those interested are referred to the special works on
the subject.'

We will first take up the simplest albuminous substances, the simple
proteins. The albumins and globulins comprise a well-characterized group.
They are generally found together; for example, in blood-serum, milk, and
the whites of eggs. The albumins are soluble in pure water. If blood-
serum is dialyzed against distilled water, a precipitate will form on stand-
ing. This is globulin, which had been held in solution by neutral salts.
Precipitation follows as the latter diffuse. The albumins, on the other
hand, remain dissolved. They are also soluble in dilute salt solutions, as

' An exhaustive description of the individual proteins is found in Otto Cohnheim'a
Chemie der EiweisskiJrper, and in Gustave Mann's The Chemistry of the Proteids, which
is based upon the former. We shall follow, a.s a rule, Cohnlieim's classification. Even
to-day the chemistry of the amino acids might be used as a basis for a cla.ssification,
tut in order to prevent misunderstandings we will here adhere to the older method.

The following works are instructive: Viktor Griessmayer: Die Proteide der Getreide-
arten, etc. flS97). Leo Morochowetz: Das Globulin des Blutfarbstoffes, etc. Le
Fhysiologiste Russe, 41-47 (190.S), and 48-60 (1904). F. Hofmeister: Ueber Bau und
Gruppierung der Eiweisskorper, Ergeb. Physiol. (Asher and Spiro), 1, 759 (1902).

132 LECTURE VII.

well as in acids and alkalies. Solutions of pure albumins are neutral.
The albumins also differ from the globulins in their behavior on " salting-
out." They are not precipitated when their neutral solution is saturated
with sodium chloride. Even saturating the solution with magnesium
sulphate solution does not produce precipitation. They are not pre-
cipitated by a half-saturated ammonium sulphate solution. In acid solu-
tions they are, however, precipitated by saturating with sodium chloride
or magnesium sulphate. The albumins, as previously stated, have been
obtained in crystalline form.

The globulins are insoluble in pure water and in dilute acids, but are
dissolved by dilute alkalies and neutral salt solutions. They can be pre-
cipitated from solution by the addition of water or acids. Passing carbon
dioxide through their solution is sufficient to precipitate them. The
globulins can, consequently, be easily coagulated. They may be redis-
solved, only when freshly precipitated. The globulins act like acids, and
turn blue litmus red. They are precipitated by a half-saturated ammo-
nium sulphate solution. They are very widely distributed. The serum-,
milk-, and egg-globulins are best known. There are, undoubtedly, other
groups of closely allied proteins, which are, at present, classified separately.
Albuminous bodies, very much like the globulins, have been isolated from
various animal organs. Thyreo-globulin * is assigned to this class. It
contains a large percentage of iodine.

The globulin-like proteins present in plant seeds are grouped separately.
They act as reserve material for the seeds, often occurring in large masses,
and are easily obtainable.^ They are occasionally found in crystalline
form, as has been mentioned, and can often be crystallized. Edestin,
the best known of these, occurs in various seeds, and can be dissolved in a
five per cent sodium chloride solution at sixty degrees, and recrystallized
therefrom. These vegetable proteins all react acid and are insoluble in
water; they, however, all dissolve in salt solutions, and can be recovered
from these by diluting or acidifying.

The phytovitellins, also called " vegetable-casein," which have been
but little investigated, are proteins obtained from plants, and are provision-
ally placed in this class. Some of them contain phosphorus, and should,
therefore, preferably, be included with the nucleo-albumins. It has not
yet been decided definitely whether this phosphorus is actually a part of
the protein molecule, or only an impurity. The latter assumption seems
justified, as the method of preparation is crude, and very little effort has
been made to purify them. The reserve proteins stored in the seeds of
various cereals belong to this class. These substances are partially
soluble in alcohol. The gluten-casein of wheat, legumin of the legumes,

' A. Oswald: Z. physiol. Chem. 27, 14 (1899); 32, 121 (1901).

' Cf. H. Ritthausen: Die Eiweisskorper der Getreidearten, etc., Bonn, 1872.

ALBUMINS OR PROTEINS. 133

conglutin of the lupins, almonds, nuts, etc., are insoluble; while gliadin,
found in wheat, rye, barley, and oats, is soluble. Zein, a protein obtained
from corn, is soluble in alcohol. This group of alcohol-soluble albuminous
bodies from plant seeds is also characterized by the absence of lysine, in
their composition, whereas almost all of the other proteins yield this as
a cleavage-product.

The group of fibrinogens and fibrin is better defined. We shall dwell
more in detail on these proteins ' later on. They have, in common with
casein and myosin, the faculty of being clotted, i.e., changed to a solid
state, by a ferment. This curdling is not identical with coagulation. Al-
though the curdled proteins are no longer soluble in water, they can still
be coagulated, i.e., completely denaturized by heating or by treating them
with alcohol. Fibrinogen is found in the blood-plasma of all inverte-
brates. It is changed into fibrin by the action of a ferment. The
coagulation of blood, which normally occurs only when the blood has left
its containing vessels, is dependent on this action. We shall see later
that this process is an extremely complicated one which has not yet been
explained entirely. •

Myosin acts very much like fibrinogen, and is found in the fibres
of striated muscle in a soluble form. Its curdling causes rigor mortis.
The cause of the curdling of this muscle material is not understood. A
ferment action, analogous to that of fibrin formation, has been offered in
explanation, although such a ferment has not yet been proved definitely
to exist. We must also mention the fact that besides myosin other
albuminous substances, among these myogen,- have been described. It
is difficult to determine whether these proteins are distinctively-charac-
terized albuminous substances, or, more probably, the same myosin in
different forms. We have seen that many proteins, including myosin,
can be very ea,sily denaturized. These products, therefore, have entirely
different properties, and easily give one the impression of containing a
protein of a peculiar nature. We cannot go far astray if we confine our-
selves, at least for the present, to calling them simply, " muscle-albumins."
We also find these same substances in the smooth muscles. Analogous
proteins must be present in other organs, as they also show the same
rigor mortis phenomenon. This, however, gradually disappeare. No
satisfactory explanation of this process has as yet been presented.

We will now discuss a group of proteins whose distinctive characteristic
is the presence of phosphorus. This group of nucleo-albumins includes
a very heterogeneous collection of proteins. They also possess another
lesser characteristic in being largely liquefied by pepsin-hydrochloric acid
digestion, the complex containing phosphorus being split off and finally

' Cf. the Lecture on Blood.

' O. von Fiirth: Ergeb. Physiol. (.-Vsher and Spiro) 1, 110 (1902).

134 LECTURE VII.

appearing as an insoluble precipitate. Later on this goes into solution.
This complex has been called paranuclein by Kossel,' and pseudonuclein
by Hammarsten.^ It is at present entirely arbitrary to include the nucleo-
albumins among the simple proteins. Didactic considerations were mainly
responsible for placing them in this class. The nucleo-albumins have
often been classified with the nucleoproteids on account of their com-
mon phosphorus content. The latter, however, are sharply distinguishable
from the former by the fact that purine bases, pyrimidine derivatives, and
pentoses enter into their composition. O. Cohnheim ' proposes instead
of nucleo-albumin the name of phospho-globulin to prevent such confusion.
This group includes casein, vitellin, and a series of cell-neucleo- albumins. It
is possible that legumin and the so-called vegetable-casein belongs to this
class. The nucleo-albumins are invariably distinct acids. When pure
they are insoluble in water. Their salts, on the other hand, are easily
soluble in alkalies and ammonia. They are precipitated by acids. Boiling
a solution of their salts is not sufficient to coagulate them.

We shall consider casein and its digestion more in detail later. Here
its occurrence in milk will be merely mentioned. Vitellin occurs in the
yolk of eggs. It has never been prepared in a pure condition. Nucleo-
albumins are supposed to occur in all cells. With the exception of casein,
no other member of this group has so far been isolated in a pure condition.
This group already shows the total inadequacy of our methods of prepara-
tion. As soon as we are compelled to attempt the isolation of a given
protein from a mixture of albuminous material by precipitation and
certain questionable reactions, we meet with unsurmountable difficulties.
The names of the various proteins are here largely derived from their
morphological source. The physical properties of the albuminous sub-
stances are naturally dependent on the medium in which they are found.
That these substances are largely influenced by extraneous conditions,
can be easily shown by studying the behavior of the same protein when
dissolved in various solvents.* The exceptionally large amounts of
admixed salts necessarily have an effect on the other physical properties
of any individually precipitated protein. Although we are undoubtedly
right in considering the albumins and the globulins as distinct individuals,
this is not the case with those others just mentioned. Many are unques-
tionably mixtures.

We shall now consider the proteins which are relatively rich in di-amino
acids. As a result of their composition they are of a more or less basic

' A. Kossel: Arch. Anat. Physiol. 1891, 181.
' O. Haramarsten: Z. physiol. Chem. 19, 19 (1893).
• O. Cohnheim: loc. cit. p. 190.

' Cf. E. Abderhalden and O. Rostoski: Z. physiol. Chem. 46, 125 (1905); E.
Abderhalden: Z. exp. Path. Therap. 2, 642, 1905.

ALBUMINS OR PROTEINS. 135

character. They are, for this reason, precipitated by alkalies, although
redissolved by an excess. They are readily soluble in acids.

The histons belong to this class. They belong just as much to the
simple proteins as to the more complicated ones. They do not occur as
such in nature. They are always linked with some other radical, and must
be separated from it when prepared for study. The first histon, in the
narrower sense, was isolated by Kossel ' from the blood corpuscles of a
goose. The histon obtained from the leucocytes of the thymus-glands
has been most carefully studied.^ The histons are very widely distributed.
They are found in the spermatozoa of fishes, and can be shown to occur
as antecedents of the protamines; for instance, in the immature testes of
the salmon.^ Many authors place globin, the protein component of hemo-
globin, in this class. It is very basic in its nature, although it otherwise
behaves differently from the other histons. It really occupies an inter-
mediate position between the histons and the simple proteins.

The histons have been very carefully examined by Ivar Bang.* He
mentions the following as characteristic reactions: — They are precipi-
tated from their water solutions by ammonia, but are redissolved by an
excess. The histons are only coagulated by boiling in the presence of
salts. They form a precipitate with nitric acid in the cold, redissolve on
heating, but again settle out on cooling. Neutral solutions of histons
give precipitates with solutions of ovalbumin, casein, or serum-albumin,
which contain but little admixed salts. This is considered a very charac-
teristic reaction. These precipitates contain one part histon and two of
casein, two of serum-albumin or one of ovalbumin. These reactions do
not apply to all histons. The various membere of the histon group differ
greatly from one another. Their chief characteristic is the large amount
of bases present.

The protamines, discovered by Fr. Miescher ^ in the mature spermatozoa
of the salmon, are closely related to the histons. A. Kossel " has greatly

' A. Kossel: Z. physiol. Chem. 8, 511 (1883-84).

' L. Lilienfeld: Md. 18, 47.3 (1894).

' Cf. F. Miescher (O. Schmiedeberg): Arch. exp. Path. Pharmak. 37, 100 (1896).
One experiment showed only about 40 per cent of bases in a product obtained in the
beginning of October from the testes of the salmon. This was evidently a mixture
of histon and protamine. A second preparation showed about 60 per cent of bases,
while the protamine obtained from mature tester showed as much as 80 per cent bases.

' Ivar Bang: Z. physiol. Chem. 27, 463 (1897); 30, 508 (1900). Hofmeister's
Beitrage, 4, 115, 331. and 362 (1903).

' Fr. Miescher: Ver. Naturfors. Gesellsch. Basel, 6, 138 (1874). J. Piccard: Ber. 7, 1714
(1874), and Fr. Miescher's complete works, loc. cit.

' A. Kossel: Z. physiol. Cliem. 22, 176 (1896); 25, 165 (1898); 26, 558 (1899);
Ber. 34, 3214 (1901); Z. physiol. Chem. 40, 311 (1903-04); also A. Kossel and A.
Mathews: ibid. 26, 191 (1898). A. Kossel and F. Kutscher: ibid. 31, 165 (1900). A. Kossel
and H. D. Dakin: ibid. 40, 565 (1903-04).

136

LECTURE VII.

extended our knowledge of them; so much so, that most of their funda-
mental constituents are already well established. Kossel and his students
have also found protamines in the spermatozoa of other fishes. They
resemble one another very closely, although they are not identical, as is
indicated by their percentages of mono- and di-amino acids. The pro-
tamine group is very well defined. They contain, above all, a very large
amount of bases. Arginine is the main cleavage-product of the protamines.
The amount varies between 58-84 per cent according to the origin of the
protamine. They also contain mono-amino, as well as the di-amino, acids,
as we shall see later. To assign to the protamines a particular position
among the albuminous substances would certainly be arbitrary and unjusti-
fiable at present. They are closely related to all the remaining proteins, and
are formed from them. There is also no justification for considering them
as the simplest proteins. Although one of the cleavage-products of the
protamines, arginine, is found in large amount, it must not be forgotten
that other amino acids are also present, and that the constitution of the
protamines may be just as complicated as that of other proteins.

The protamines can be purified, which is of great advantage in their
preparation. The free protamines are obtained pure only with difficulty.
They are best obtained in the form of sulphates, and then changed over
into chlorides. The latter can be precipitated from methyl alcohol solution
by means of platinum chloride. M. Goto ' has analyzed these platinum
salts, and obtained the following values:

Salmine (from salmon) . . .
Clupein (from herring) . . .
Scombrine (from mackerel)
Sturine (from sturgeon) . .

c

H

N

Pt

CI

22.96

4.32

14.83

24.73

26.56

22.81

4.30

12.59

24.64

26.57

23.49

4.75

13.57

24.09

25.99

24.32

4.49

14.20

23.10

25.42

6.70
9.09
8.11
8.47

Sulphur has not yet been discovered in the protamines, and is probably
absent. We are not at present aware of any reason why a lower
molecular weight should be assigned to them than to the other proteins.
The protamines are not coagulated by heat. While the ordinary proteins
are precipitated by the alkaloid reagents (for instance, phospho-tungstic
acid) only in acid solution, and the histons in neutral solution, the prot-
amines will be even thrown out in alkaline solution. The protamines can be
salted-out by ammonium sulphate and sodium chloride.

The protamines have toxic properties; ^ 15-18 mg. of scombrine, salmine,

' M. Goto: Z. physiol. Chem. 37, 94 (1902).

» W. H. Thompson: Z. physiol. Chem. 29, 1 (1900).

ALBUMINS OR PROTEINS. 137

or clupein, and 20-25 milligrams of sturine, per kilo weight of the animal,
are sufficient to kill a dog. It is still undecided whether this poisoning is
due to the protamines or to some admixture.

Besides the protamines mentioned, cyclopterine ' from the lump-fish,
{jC yclopterus lumpus), and acipenserinc, from the testes of the sturgeon
(^Acipenser stellatus),- are also described as protamines. Two other pro-
tamines may be mentioned: a- and ^-cyprinine,^ which are obtained from
the sperm of the carp {Ciprinits carpio). The sperm of the brook-trout
(Salmo fario), the white snapper {Coregonus oxijrhynchus), the sheath-
fish {Silurus glanus), and the pike {Esox lucius),* also contain protamines.
Protamines have not been isolated positively from any other representa-
tives of the animal kingdom. The significance of the protamines has not
yet been established.

Related to the proteins just described is a group of albuminous bodies,
whose biological significance differs materially from that of any so far
mentioned. Their common properties have united the heterogeneous
substances into one group, called the " albuminoids." They constitute
the frame-work of the animal tissues. The)- are not found in the cell pro-
topliism, nor in the tissue fluids. We shall see later, that their significance
also corresponds to their entire composition. They are not to be considered
as nutrient materials in the narrower sense, and participate but little in the
intermediate metabolism. Incidentally they are difficultly digestible;
in fact, being somewhat resistant to the digestive ferments. The albu-
minoids are to the animal body what the higher carbohydrates (for
instance, cellulose) are to the vegetable. They are all insoluble in
water and salt solutions. They are only slightly attacked by acids and
alkalies. It is practically useless to attempt to purify the albuminoids.
They can only be studied in the manner in which they occur in nature.
It is an entirely arbitrary assumption that they exist as a chemical
entity.

Collagen occupies a special position among the albuminoids. It con-
stitutes the foundation of the bones and cartilages, and constructs the
fibrils of the connective tissues. • It may be extracted from these tissues
by boiling with water. The product which goes into solution is called
glue, glutin, or gelatin. In contra-distinction to the other proteins,
collagen is soluble in warm water, and solidifies on cooling. Numerous
investigations indicate that it is not an individual substance, undoubtedly
differing according to the animal, or organ, from which it is obtained.
The nature of the change underlying the formation of gelatin has

' N. Markowin: Z. physiol. Chem. 28, .31.3 (1899).

' D. Kurajeff: Z. physiol. Chem. 32, 197 (1901).

' A. Kossel and H. D. Dakin: i6i</. 40, 5Go (1903), he. cit.

* A. Kossel: ibid. 22, 17G (1896), loc cit.

138 LECTURE VII.

been but little studied. It may possibly be due to a hydrolytic decom-
position.

Another group of albuminoids is included under the name of keratins.
They comprise the so-called " horn substance," and are found, as such,
in the hair, feathers, nails, hoofs, horns, etc. N euro-keratin, which forms
a part of the sheath of the medullary nerves, belongs to this class. The
keratins are noted for their insolubility in water, dilute acids and alkalies.
They have a high percentage of sulphur. Ovokeratin, in the membranes
surrounding the eggs of many animals, is closely related to the keratins.
Gorgonin, constituting the foundation of the axial skeleton of coral
{Gorgonia cavolini), is also included. It is especially noted for its high
percentage of iodine.

The elastin bodies form a group by themselves. They predominate in
the formation of the elastic tissues. Elastin is generally prepared from
the Ligamentum nuchce of the ox.

Fibroin, obtained from the threads of silk-worms, is the best known
albuminoid. It is characterized by a very low percentage of di-amino
acids. We shall return to this substance later.

Spongin, forming the framework of sponges, and conchiolin, the basis
of the skeleton of the mussel, are included among the albumoids. Amy-
loid, to whose content of chondroitin-sulphuric acid we recently referred,
is also assigned to this group. It occurs only under pathological condi-
tions. It is found, on the one hand, in the form of small kernels in the
brain (the so-called Corpora amj/lacea), and then, again, as large deposits
in the parenchyma of many organs. This is called an amyloid degenera-
tion. The cause of its formation is but little known.

We must finally consider the group of the albumoids. It includes
the most varied albuminous substances, about whose constitution nothing
is at present known. To this class jjelong the Membranw proprice of many
glands, sarcolemma, osseo-albumoid, chondro-albumoid, the albumoid of
lentils, the elementary matter of Chorda dorsalis, ichthylepidin (occurring
in the scales of fishes), the horny layer in the crop of birds, reticulin (which
composes the reticular tissues of the intestinal mucosa), and many other
analogous substances.

It is impossible, at present, to give any further exact account of the
various representatives of the albumoids. The fact that they are
obtained with difficulty, and, more especially, that it is almost impossible
to purify them, has made any exact study of the class up to the present
time entirely futile. Observers have concerned themselves chiefly with
an investigation of various physical properties of individual proteins of
this group, and especially to the action of ferments upon them. Their
practical indigestibility, has made it possible to remove the ordinary pro-
teins from them. The great predominance of albuminous substances, not

ALBUMINS OR PROTEINS. 139

only in the cells, but also in all the fundamental and basic elements, dis-
tinctively separates the animal from the vegetable world. As the plants
are capable of utilizing the very diverse carbohydrates in building up
the various polysaccharides, so the animal organism is able to assimilate
the simple proteins (those of milk, for e.xample) for its nourishment, pro-
ducing the large number of albuminous substances found in the cells and
tissues. The albuminoid and albumoid groups are good illustrations of
how the animal organism utilizes the proteins for its varied functions, and
how it adjusts the composition to the function.

We shall now discuss the proteids, or compound proteins. We can
do this rather rapidly, because the non-albuminous component of the
proteids is the more interesting, and will be considered elsewhere. The
large number of nucleoproteids belong to this group. They are com-
posed of albumin and nucleic acid. The former may consist of certain
of the protein groups previously mentioned. We are acquainted with
compounds of nucleic acids with histons, as well as with protamines. It
is an open question as to whether other proteins do not likewise partici-
pate in the formation of nucleoproteids. It must be admitted, how-
ever, that the very existence of the nucleoproteids has been questioned.*
Nucleic acid has the property of precipitating protein from solution. In
preparing the nucleoproteids we only extract the various organs with,
for instance, water. On adding acid, generally acetic acid, the nucleo-
proteids are precipitated from the extract. We can imagine that the
nucleic acid is present in the extract perhaps in the form of its sodium
salt, together with the albumin. As a matter of fact, by acidifying, the
nucleic acid is freed, which precipitates the soluble albumin. We can, by
adding nucleic acid to albuminous substances, obtain precipitates which
are very much like nucleoproteids. Nucleoproteids have, however, also
been obtained by " salting-out." - It is, therefore, very probable that
the nucleoproteids are present, as such, in the various organs.

We do not, at present, know the manner of linking between the nucleic
acid and the protein. There are apparently various kinds of combina-
tion. For instance, 0.8 per cent of hydrochloric acid is sufficient to
separate histon from the nucleo-histon obtained from the thymus gland;
on the other hand the pancreas-proteid is decomposed into nuclein and
albumin, even in neutral solution on boiling. The composition is not as
simple as the name nucleoproteid might indicate. There seems to be more
than a mere combination of nucleic acid and protein. If a nucleoproteid
is decomposed, we, of course, obtain a protein, for instance histon. The

' I. Bang: Z. physiol. Chem. 30, 508 (1900). T. B. Osborne and I. F. Harris: ibid.
38, 85 (1902).

' F. Malengreau: La Cellule, 17, 339 (1900). W. Huiskamp: Z physiol. Chem. 32,
145 (1901); 39, 55 (1903).

140 LECTURE VII.

other compound is not a nucleic acid, but a combination of tliis with
albumin. This is called nuclcin. On further decomposition, it breaks
down into albumin and nucleic acid.

The nucleoproteids are all soluble in water and salt solutions. They
are very soluble in alkalies. They are distinctly acid in their charac-
teristics. They are precipitated by acids, although redissolved by an
excess. The nucleoproteids can be " salted-out " and coagulated by
heat and other means. When nucleoproteids are digested with pepsin-
hydrochloric acid, nuclein settles out, while the albuminous cleavage-
product is dissolved by the ferment in the usual manner. Fr. Miescher,'
the discoverer of the nucleins, noticed this characteristic property. The
nucleins themselves are but little affected by pepsin-hydrochloric acid,
although more so by trypsin. It is very difficult to purify them ; in fact, it
is doubtful whether they have ever been isolated in a pure condition.

The nucleoproteids often contain iron, and it is very probable that the
main supply of this element in the system occurs in these, and in hemo-
globin. They are present in every cell, and are found in the nucleus.
Miescher first noticed them in the little pus cells. They were isolated
shortly afterwards also from the blood corpuscles of birds and snakes.
We must also state that efforts have been made to place the ferments
themselves in the group of nucleoproteids. By the discovery of nuclein
and the nucleoproteids, Fr. Miescher has substantially advanced our
knowledge of the constituents of the nuclei. The fact that all nuclei —
•whether plant or animal — contain nucleoproteids is another link between
these two great kingdoms. We must not, however, forget that we know
practically nothing about the biological significance of these substances.
They have interested us greatly, especially on account of their predomi-
nance in the nuclear material, and the ease of obtaining them. The
importance assigned to them may, however, be entirely unwarranted.
Our knowledge of the other constituents of the nuclei is far too meager
to tell us much concerning the functions of the nucleus.

Nucleoproteids have been isolated from almost every organ. So, also,
from the spermatozoa-masses. Those of fishes contain up to 96 per cent
of nucleic-acid-protamine, or -histon. Fr. Miescher and Schmiedeberg give
the following composition to salmon spawn: 60.5 per cent nucleic acid,
35.56 per cent protamine. A nucleic-acid-histon has been obtained from
the sea-urchin {Arhacia pustulosa).- The spermatozoa of bulls also
contain a nucleoproteid, which, however, does not contain protamine
nor histon, but some other kind of protein.' Nucleoproteids have also
been isolated from the thymus gland, from the red corpuscles of birds

' Fr. Miescher: Hoppe-Seyler's Med.-chem. Untersuchungen, p. 441 (1871).

' A. Mathews: Z. physiol. Chem. 23, 399 (1897).

' Fr. Miescher: Arc. exp. Path. Pharm. 37, 100 (1896).

ALBUMINS OR PROTEINS. 141

and reptiles, from the pancreas, from the gastric juice, from the thyroid
gland, from suprarenal glands, and from muscles. Nucleoproteids have
also been found in tumors. The nucleoproteid of yeast has been much
studied on account of the ease with which its nucleic acid is obtained.
We must also call attention to the presence of nucleoproteids in the
vegetable kingdom.

Oxy-hemoglobin likewise belongs to the group of proteids. It is
composed of globin and hematin. We have met the former while dis-
cussing the histons. Oxy-hemoglobin contains, according to the inves-
tigations of Fr. N. Schulz,' about 4-5 per cent hematin. We do not, at
present, know whether different kinds of animals possess different kinds
of hemoglobin; in fact, we are not at all certain that one and the same
species of animal has a uniform hemoglobin. The crystalline form of the
hemoglobin is of little value. The investigations of the globin portion
have also been of little service. The hemoglobin from the horse has been
most thoroughly studied. The decomposition of the hemoglobin from
the dog gave corrresponding amounts of amino acids. The second com-
ponent, hematin, seems to have constant properties, irrespective of the
animal from which it is obtained. We shall devote more attention to
hematin when we discuss the composition of blood. We shall also have
to consider then the part hemoglobin plays as a carrier of oxygen and
carbon dioxide.

The glucoproteids form the third subdivision of the proteid group.
They are composed of a protein and a carbohydrate complex. We have
already met them in our discussion of the carbohydrates,^ and have
seen that glucosamine is produced by the hydrolysis of the ordinary
mucins, while galactosamine is obtained from the mucin of frog-spawn.
These are the only carbohydrates that have been positively isolated from
the glucoproteids. It is, indeed, very questionable whether the carbo-
hydrates mentioned pre-exist as such. The whole group of glucoproteids
is still very indefinite. While it is comparativelj' easy to decompose the
nucleoproteids and hemoglobin into their albuminous and non-albumi-
nous constituents, this does not hold with the glucoproteids. The carbo-
hydrate group is only split off by boiling with mineral acids or by the
action of alkalies. It is, of course, possible that the glucoproteids are
true albuminous bodies, differing only from the other proteins, in that
more carbohydrates take part in their formation. Certain of the ordi-
narj^ albumins may indeed really belong to this group. If this be true,
we would then Iiave all shades of proteins, some with considerable car-
bohydrates others with less, and some which do not contain any carbohy-
drate at all in the molecule.

' Fr. N. Schulz: Z. physiol. Chera. 22, 449 (1898).
' a. pp. 20 and 25.

142 LECTURE VII.

It would be much more correct if we, for the present, entirely drop
the name glucoproteid until we were certain that the carboliydrates
occupy the same relative position to the protein in the molecule, as, for
example, hematin does to hemoglobin. We will, therefore, classify the
glucoproteids as true proteins, in the largest sense, and deal with the car-
bohydrate cleavage-products in the same manner as with the other pro-
tein components. It is certainly a matter of considerable interest to know
that amido-carbohydrates — that is, sugars which are intermediate be-
tween the amines and the true carbohydrates — take part in the building
up of these proteins.

To this group belongs a series of albuminous substances whose physical
appearance is sufficient to identify them as a class. They are called
mucins and rmicoids. They may be recognized even by their elementary
composition. The occurrence of the carbohydrate groups rich in oxygen
lowers the percentages of carbon and nitrogen. The amount of carbo-
hydrate present is a very variable one, ranging from 3-37 per cent, accord-
ing to the substance in question. It is very difficult to purify them even
approximately. They are not coagulated by heat, — a property which dis-
tinguishes them from the ordinary proteins. They may, nevertheless, be
easily denaturized. They can be " salted-out." The mucins and mucoids
are distinctively acid, and can be precipitated by acids. They are readily
soluble in alkalies, alkaline carbonates, and ammonia.

The mucins are very widely distributed. They constitute the slimy
material of many secretions, and are eliminated by the respiratory ' and
digestive tracts, sometimes from individual cells (goblet cells) and again
from larger glands — such as the salivary glands. There are also mucous-
producing cells in the bile ducts and the urinary passages. The mucins are
also produced to a consideraljle extent by invertebrates, e.g., slime of snails.
The mucin from the respiratory passages ' and from the submaxillary
glands,^ has been studied most. Mucin from the invertebrates does not
seem to be excreted as such, but is only produced secondarily from a sub-
stance called mucinogen.

Proteins closely related to the mucins have been observed in ovarian
cysts, which are peculiar tumorous formations of the ovaries. They are
called para- and pseudo-niucin.* The latter differs from the ordinary
mucins in that it is not precipitated from its solutions by acetic or nitric
acids. Paramucin is occasionally found in gelatinous masses in cysts. It
resembles mucin in being precipitated by acids.

The mucoids are closely related to the mucins. They are found to

> F. Muller: Z. Biol. 42, 468 (1901).
' O. Hammarsten: Z. physiol. Chem. 12, 163 (1887).
' O. Hammarsten: Pfliiger's Arch. 36, 373 (1883).
* O. Hammarsten: Z. physiol. Chem. 6, 194 (1882).

ALBUMINS OR PROTEINS. 143

some extent dissolved, and to some extent they participate in the con-
struction of tissue. Their classification is based upon principles morpho-
logical rather than chemical-physical. They are sometimes included with
the mucins, and sometimes classed as an independent group. We shall
mention only the most important of them. Deserving of mention are the
mucoids prepared from tendons, bones, and cartilages. The last named
has been thoroughly investigated. Chondro-miicoid in conjunction with
collagen constitutes the elementary material of cartilage. It contains
considerable sulphur and a reducing sugar. When hydrolyzed we obtain
a protein and a carbohydrate-ethereal sulphuric acid, the so-called chon-
droitin-sidphuric acid} This is a colloidal substance, and has been investi-
gated by Schmiedeberg,^ and later by A. Orgler and C. Newberg.' By
boiling with acids for a short time it decomposes into sulphuric acid and
a residue, chondroitin, free from sulphuric acid. Further treatment with
acid produces an amido-polysaecharide, whose exact nature has not yet
been determined. Chondroitin-sulphuric acid is found not only in carti-
lages, but also in bones in ligamentum nuchce, and in the mucous mem-
brane of the pig. It occurs especially in amyloid, a protein which is
found in the tissues under certain pathological conditions. Morner ■*
found chondroitin-sulphuric acid repeatedly in the urine to the extent of
0.05 per cent.

The proteins found in the vitreous humor, in the cornea, and in the um-
bilical cord, also belong to the group of mucoids, as does the ovomucoid
obtained from the white of an egg. The latter may be isolated by coagu-
lating the globulin and albumin, and adding alcohol to the filtrate. A
reducing substance, gluocsamine, can be split off from it. Steudel ^ ob-
tained 29.4 grams glucosamine from 100 grams ovomucoid. Blood serum
also contains a mucoid body. Morner describes another substance be-
longing to this group, which he obtained from the urine. An analogous
product also occurs in the ascitic fluid.

We must admit that a large amount of uncertainty attaches to this
group of albuminous substances. Secure foundations are lacking. It is
practically impossible to purify these products, beyond getting rid of
gross impurities. The properties of the various members of this group
are such, that it is very difficult to work with them. It is diflacult to ob-
tain them in large quantities. We must, therefore, look with skepti-
cism upon all new mucins and mucoids, and should await the time when
purely chemical investigations will have sufficiently classified the mem-

> C. T. Momer: Skand. Arch. f. Physiol. 1, 210 (1889). Cf. Lecture III, p. 49.
' O. Schraiedeberg: Arch. exp. Patli. Pharm. 28, .355 (1891).
' X. Orgler and C. Neiiberg: Z. pliy.siol. Chem. 37, .399 (1903).
• K. A. H. Morner: Skand. Arcli. Physiol. 6, 3.32 (1895).
» H. Steudel: Z. physiol. Chem. 34, 353 (1901-02).

144 LECTURE VII.

,bers of this group, at least to the extent of recognizing the nature of the
individual decomposition products.

Hammarsten ' has isolated a peculiar substance from the albuminous
gland of the Roman snail {Helix pomatia), which is possibly a protein,
difficult to classify, or, more probably, a proteid. It contains a liEVO-
rotary carbohydrate, which yields a dextro-rotary substance on boiling
with acids. This material also contains phosphorus. It does not belong
to the nucleo-proteids, because it possesses no xanthin bases. To this
group, called " phospho-glucopjoteids," is assigned the substance ichtulin,
which has been obtained from fish eggs.

The proteins and proteids already mentioned do not, by any means,
exhaust the list. We have mentioned only those which have been fairly
well characterized, and which are starting points for further investiga-
tion. The albuminous substances, and especially some of the albumin-
oids, isolated from plant seeds and the animal fluids, are undoubtedly best
known. We frankly admit that our knowledge is very vague concern-
ing the albumins which participate in the construction of the various
organs. It is very probable that the number of proteins and proteids
occurring in the tissues is extremely large as is indicated by the descrip-
tion of the various tissue-albumins. On the other hand, we must remem-
ber that when proteins are attacked but slightly their properties are
changed completely so that they may be mistaken for new kinds of pro-
tein. It is also probable that the tissues, and especially the cell proteins,
are continually undergoing changes. It is still undecided whether the
albuminous constituents of the tissues are to be considered as stable sub-
stances, to a certain degree, or as undergoing continual decomposition
and reconstruction. From this question arises the extremely important
problem of the whole subject of protein metabolism, about which we are
still in the dark.

We have, so far, neglected to mention a group of nitrogenous com-
pounds, which are closely related to the proteins. These are the
melanins, which are very widely distributed in the animal kingdom.
They are found in the very large number of pigments occurring in the
hair, feathers, choroidea of the eyes, skin, etc. Their presence in tumors
is very interesting; and their unusually large occurrence in the melano-
sarkoma of horses — especially of white horses — has drawn much atten-
tion to them. In the muscles of these animals (for instance, the glutsei),
there are often embedded very large tumors, which appear, according to
age, as very black, firm masses, or like a cyst, containing an inky, finely
granulated fluid. It is certainly significant that these masses of pigment
are obtained in animals whose hair has no pigments. The pure white

O. Hammarsten: loc. cit. Pflijger's Ann. 36, 373 (1883).

ALBUMINS OR PROTEINS. 145

horse produces, therefore, a pigment, but it is not utilized. This pigment,
called hippomelanin, has been very exhaustively investigated.' It occurs
. as a very finely divided, brownish-black powder. No individual constit-
uent of melanin has so far been definitely isolated. The constituents of
the other melanins, prepared from the hair, choroidea, etc., has also never
been cleared up. The melanins are, undoubtedly, not simple substances.
It is difficult to obtain any exact knowledge about these substances,
because they are not readily purified. They are extremely resistant to
acids and alkalies, and to oxidation and reduction processes. Some will
dissolve in alkali; others will not. Some contain iron; in others, this ele-
ment is lacking. It has been suggested that, because some of the melanins
contain iron, they are related to the coloring matter of the blood. It
is possible that some of these pigments may trace their origin back to
hematin, although no proof has been presented to substantiate this
hypothesis. The melanins are characterized by a high carbon and a low
hydrogen content. Many of them have considerable sulphur in their
composition.

By the hydrolj'sis of almost all the albuminous bodies with acids,
products are obtained which very much resemble the naturally-occurring
melanins. These black products are called humin substances. They are
supposed to be related to the natural melanins, and the suggestion has
been made that glucosamine, trj'ptophane, tyrosine, and lysine participate
in the formation of pigment. We know nothing definite - about the
constituents of these humin substances, and are unable to decide whether
there is any distinct relationship between these and the melanins. We
will particularly emphasize the fact that the hypothesis just mentioned
to the effect that the humin substances are built up of the fundamental
constituents of the albumins, is entirely without empirical justification.

We have now mentioned the most important classes of the remarkably
diversified group of the proteins, or albuminous substances. The unsatis-
factoriness of the whole system of classification is evident from our pre-
sentation of the subject. It only .serves as a temporary basis for further
orientation. It is highly important that we should sharply and distinctly
emphasize how slight the proofs are in the case of most of these substances
that we are dealing with simple substances rather than with mixtures,
and how extraordinarily cautious we must be for this reason in passing
judgment upon the results, which must eventually be referred to more
physico-chemical investigations.

' J. Berdez ami M. Nencki: .Arch. exp. Path. Pharm. 20, .346 (1885). M. Nencki
and N. Sieber: ihid. 24, 17 (1887).

' Cf. also Fr. Samuely: Hofmeister's Beit. 2, 355 (1902)

LECTURE VIII.

ALBUMINS OR PROTEINS.
II.

The Components of Protein.

Although the number and distribution of the proteins in Nature are
very great, still they show great similarity in the way they are constructed.
Various methods have been tried for effecting the cleavage of proteins.
Up to the present time, hydrolysis, whether brought about by the action
of acids, of alkahes, or of ferments, has alone been productive of results.
Experiments performed in the attempt to obtain known products by
oxidation have thus far been unfruitful.' It is, moreover, hardly to be
expected that in the case of such a complicated substance as albumin we
shall obtain much idea of its chemical constitution by means of oxidation
or reduction processes. We shall limit ourselves here, therefore, to a
consideration of those investigations which have been of service in the
further development of the entire chemistry of the proteins. We shall
first of all take up those substances which are formed lay the hydrolysis
brought about by the action of acids or alkalies. If a protein is boiled
for some time with fuming hydrochloric acid, or with twenty-five per
cent sulphuric acid, its character is completely changed. It is broken
down into numerous, simpler cleavage-products. These are of various
kinds. They possess, however, certain common characteristics. As far
as we know, they nearly all crystallize well, and contain nitrogen, hydro-
gen, and oxygen. These cleavage-products are known in general as amino
acids. They can be easily identified and prepared in a pure condition.
Besides these amino acids we find varying amounts of ammonia, and fre-
quently humin substances. We shall presently see that we are acquainted
with a large number of the cleavage-products of the proteins, although an
appreciable part of the molecule is still unexplained. We shall also
soon learn that all proteins, as far as our present knowledge goes, contain
these same amino acids. Occasionally one or another amino acid is
missing, although, as a whole, there is a very general agreement in the
qualitative composition. The following amino acids have been isolated
up to the present time:

' Cf. Otto von Fiirth: Hofmeister's Beitrage, 6, 296 (1905). The older literature is
carefully compiled in this article.

146

1. Moiio-amino-mono-carboxylic acids.

ALBUMINS OR PROTEINS. 147

I. ALIPHATIC SERIES.

GlycocoU.
Alanine.

Amino-isovaleric acid.
Leucine. -n - oi/wwa-"

Isoleucine. r-\ ^._.....

2. Mono-amino-hydro.xy-mono-carboxylic acids: Serine.

3. Mono-a.nino-di-carbo.xylic acids . . I Aspartic acid.

I Glutamic acid.

4. Di-amino-carboxylic acids J ^''

[ .\rginine

5. Di-amino-hydroxy-mono-carboxylie acids. Di-amino-tri-hydroxy-dodecylic acid.

6. Amino acids containing Sulphur: Cysteine and Cystine.

n. AROMATIC SERIES.

Mono-amino-mono-earl)oxylic acids: Phenylalanine.
Mono-amino-hydroxy-mono-carboxylic acids ; Tyrosine.

ni. HETEROCYCLIC COMPOUNDS.

Monoa-amino-mono-carboxylic acids . \ Pyrolidine-carboxylic acid.

( Tryptophane.

Hydroxy-mono-amino-inono-carboxylic acids: pyrrolidine-carbo.xylic acid.

Histidine.

The carbohydrate group should also be included. These occupy a pecu-
liar position, because they are absent from a large number of the proteins;
in others their occurrence is questioned; while in still another group of
proteins they appear in larger amount, but only in part as a direct constitu-
ent of the albumin molecule. Many authors classify all these proteins con-
taining carbohydrates as compound albumins. As previously indicated,
we do not consider this as justifiable. We shall subsequently return to
this carbohydrate group.

Let us turn to the individual amino acids. We shall consider their
distribution when we return to the composition of the individual proteins.
For the moment we will only classify them according to their constitution,
as this is necessary in order to understand their biological significance.

The mono-amino-mono-carboxylic acids can be derived from the normal
fatty acid scries: CnHanOo. The simplest member of this series is glyco-
coll, also called glycine or amino-acetic acid:

CHsCNHo)'

COOH

' We shall use this formula, but the following is also possible:

COO

CH -XII,

148 LECTURE VIII.

GlycocoU was one of the first-known cleavage-products of proteins. As
early as 1820 Braconnot ' obtained it, in conjunction with leucine, on boiling
glue with dilute acid or alkali. It is also obtained, as such, from the
muscles of the scallop, Pecten irradians.

Alanine is the next homologue of glycocoll. It is an a-amino-propionic
acid: CH3 . CH(NH2). COOH. It contains an asymmetric carbon atom,

*, and is consequently optically active, as are most of the other acid
cleavage-products of albumin, which itself rotates the plane of polarized
light. Alanine, as it occurs in nature, is dextro-rotary.

An amino-butyric acid has been described as a cleavage-product from
proteins. Later investigations, however, have not shown its presence in
the protein molecule. On the other hand, its next homologue, amino-
valeric acid, has been obtained very often. The amino-valeric acid so far
isolated from the proteins does not have a normal chain, but a branch-

ing one. It is an «-amino- isovaleric acid: pTT^>CH. CH(NH2)C00H.

LXI3 ^

It is dextro-rotary.

Leucine also has a branching chain, and is an a-amino-isobutyl-aceticacid:

pJj3>CH . CH2 . CH(NH2)C00H. The leucine, usually obtained by the

cleavage of proteins, is Z-Ieucine. In this form it occurs in many plants
and in invertebrates. Penicillium glaucwm produces rf-leucine from the
inactive leucine. The constitution of leucine has been proved by E.
Schulze and A. Lickiernik.^

Felix Ehrlich ^ has recently separated an isomer of leucine from molasses
sludge, and, soon after, also showed its presence in many plant, and
animal proteins. It is an «-amino-methyl-ethyl-propionic acid:

r H^>CH . CHNH2 . COOH.

Its constitution has been proved by its synthesis, and also that it is
decomposed by pure-culture yeast into rf-amyl-alcohol. The relations
of iso-leucine to d-amyl-alcohol are shown as follows. We will com-

' H. Braconnot: Ann. Chira. Phys. 13, 113 (1820).

2 Ber. 24, 669 (1891), and Z. physiol. Chem. X7, 51.3 (189.3).

* Felix Ehrlich : Ber. 37, 1809 (1904) ; Z. Ver. Zuckerind, 1904, 975 ; 55,
592 (1905). We shall dwell upon the syntheses and decomposition of the amino
acids, only as much as is necessary in order to understand biological processes.
We may also add, that this form of synthesis has often been utilized in the reac-
tions between ammonia and the halogen fatty acids. Cf. E. Fischer: Ber. 39, 530
(1906).

ALBUMINS OR PROTEINS.

149

pare at the same time the formation of ordinary leucine from iso-amyl-
alcohol:

CH;

CH

3>CH. CHo. CH2OH

iso-amyl-alcohol
+ O
- H2O

J^"JJ^>CH.CH2. OH

UoXls

I d-amyl-alcohol
+ 0
- H2O

i

CH;
CH;

3>CH.CH2. CHO

iso-valeraldehvde
+ HCN + NH3
- H.,0

i^S^>CH.CHO

I d-valeraldehvde
I + HCN + NH3

H.O

^JJ''>CH . CH2 . CHNH, . CN

I iso-valero-amino-nitril
+ 2H2O
- NH,

i

p^„-^>CH.CHNH,.CN
02n.5

1 d-valero-amino-nitril

+ 2 HoO

•'■ - NH3

CH
CH

3>CH . CH2 . CHNHo . COOH

a-amino-isobutyl-acetic acid

= leucine

JJJj3>CH . CHNH2 . COOH

ci-amino-methyl-ethyl-propionic
acid
isoleucine

Serine is closely related to alanine. It was isolated in LS6.5, by Cramer,*
from silk glue (or sericin). It is an a-amino-/3-oxypropionic acid:

CH2(0H). CH(NH2). COOH.O. Serine as it occurs in nature is Iffivo-

*
rotary.^

Of the di-hasic amino acids only two are known: a-asparti<: acid and
glutamic acid. The former is an a-amino-succinic acid.

COOH

I
*CH(NH2)

I
CH2

I
COOH.

' E. Cramer; J. prakt. Chom. 96, 7G (180.5).

' For the synthesis from ammonia, hydrocyanic acid, glycolaldehydc, cf. E. Fischer
and H. Leuchs: Sitzher. .\kad. Wiss. Berlin, 1902, and Ber. 35, .3787 (1902).
' Emil Fischer: Ber. 40, l.iOl (1907).

150 LECTURE VIII.

The latter is the next higher homologue, a-amino-glutaric acid:

COOH

I
*CH(NH2)

I
CH2

I
CH2

I
COOH.

Although the amino acids previously mentioned which possess both car-
boxyl and amino groups are not distinct acids nor bases, but possess their
combined characteristics, these dicarboxylic acids have a distinctively acid
character. The natural aspartic acid is the Isevo-rotary. Glutamic acid
rotates polarized light to the right. Both dicarboxylic acids occur widely
distributed in the vegetable kingdom as amides. Thus asparagine is an
amide of amino-succinic acid:

CONH2

I
*CHNH2

I
CH2

I
COOH

Asparagine.

It was first found in asparagus sprouts. Soon afterwards it was found
that the asparagine collects in the embryo, which are kept in the dark.
Asparagine seems to play an important part during germination. E.
Schulze,^ whom we have to thank for very exhaustive investigations
on the accumulation of asparagine in embryo, gives the following values:

Age of embryo in days . . 4 7 10 12 15 16

% asparagine of the dry sub-
stance of the embryo . . 3.3 11.2 17.3 22.3 25.0 25.7

% asparagine of the dry
substance of the seeds

As regards the distribution of asparagine in various portions of the
embryo, it is worthy of note that Schulze found 31 .81 per cent of aspara-
gine in the dry substance of the axillary organs of lupines, while the
cotyedons gave only 7 . 62 per cent. Asparagine occurs in plants in the
dextro- and Ijevo-rotating varieties. The latter occurs more abundantly.

4

7

10

12

15

3.3

11.2

17.3

22.3

25.0

3.12

9.78

15.24

18.22

19.43

E. Schulze: Landwirtsch. Jahrb. 1878, 411.

ALBUMINS OR PROTEINS. 151

The two varieties can be easily recognized by their crystalHne structure.
One is left-handed hemihedral, and the other right-handed. The d-vaviety
tastes sweet, while the other is insipid.

Glutamine, the amide of amino-glutaric acid, has been found in germi-
nating pumpkin seeds, by Schulze and Barbieri.' Its occurrence is simi-
lar to that of asparagine.

As far as we know, these two amides do not occur in the animal organism.
We shall later on consider their value as a food material.

Before discussing the properties of the di-amino acids, we shall devote
a little attention to the remaining mono-amino acids. Phenylalanine, first
discovered by Schulze and Barbieri ' in the embryo of lupines, has recently
been recognized as an invariable constituent of all the albumins so far
investigated. Its constitution is that of phenyl-amino-propionic acid:

CsHs. CH . CHNH2COOH. It occurs in nature as the ^variety.
*
Another aromatic amino acid, which has been known for a long time
as a constituent of albumin, is tyrosine, which can, be easily isolated on
account of its difficult solubility in water. It is p-hydrpxy-phenyl-

amino-propionic acid: C6H4OH . CUo . CHCNHo) . COOH. It occurs in

*

nature in both modifications, although mainly in the laevo form.

Tyrosine gives several color reactions, of which that of Hoffmann is the
most important. It is known under the name of MiUon's reaction. If
tyrosine is boiled with a nitric acid solution of mercuric oxide, containing
a little nitrous acid, the liquid becomes colored, and the resulting precipi-
tate is rose-colored or a dark brownish red. This reaction is not confined
to tyrosine. It is given by all benzene derivatives, in which a hydrogen
atom has been substituted by a hydroxyl group. This reaction has
become of great importance on account of the fact that all proteins
which contain tyrosine will give it. Millon's reaction is, therefore, a test
for proteins.

We now come to the heterocyclic compounds. Two representatives of
this class have been discovered by Emil Fischer:^ a-pyrrolidine-carboxylic
acid, also called proline, and hydroxy-pyrrolidine-carboxylic acid; the latter
being probably an hydroxy-(x-pyrrolidinc-carboxylic acid:

CHo— €H,

I I and C5H9NO3.

CHo *CH . COOH
\ /
NH

Proline Hydroxy-proline

' E. Schulze and J. Barbieri: Ber. 10, 199 (1877).
' Iliut. 14, 1785 (,KS81); Z. f. physiol. Cliem. 12, 405 (1888).

» E. Fischer; Z. physiol. Chera. 33, 151 (1901); Ber. 35, 2660 (1902). Cf. H. Leuchs:
Ibid. 38, 19.37 (1905).

152 LECTURE VIII.

Both proline and oxy-proline have been found in the decomposition
products of most albumins. The first is invariably present. It is, of
course, a question whether we are dealing with primary products. It is
possible that they are produced secondarily from another substance by
ring formation. Sorensen ' has suggested that the primary substance in
the production of proline may be a-amino-o-hydroxyvaleric acid. We
have not yet succeeded in isolating the amino-hydroxyvaleric acid, and
it must be remembered that a-proline is obtained by alkali hydrolysis,
as well as by acid.^ A small amount of proline has also been obtained
by peptic ^ and tryptic * digestion. We have nothing at hand at present
to warrant us in excluding a-proline and oxy-proline as primary albumin
decomposition products.

Tryptophane also belongs to this class of amino acids. It had been
known for a long time, that especially in tryptic digestion mixtures ° some
substance was present which was characterized by certain color reactions.

In an acetic acid solution it gives with chlorine or bromine water a violet
color. Also, if we insert a pine splinter, which has been previously dipped
into hydrochloric acid and then rinsed off, into a concentrated tryptophane
solution, the stick will turn purple on drying. This is the so-called pyrrole
reaction. It was soon shown that many of the reactions characteristic of
the albumins were due to the presence of tryptophane. If we add a little
glyoxylic acid, and then concentrated sulphuric acid, to a water solution
of albumin, a beautiful blue-violet coloration appears. O. Neubauer and
Rohde ° have indicated recently a new reaction for tryptophane in albumin.
If we add to a water solution, or suspension, of albumin, 5 to 10 drops of a
5 per cent solution of p-dimethyl-amino-benzaldehyde containing 10 per
cent of sulphuric acid, and then, while shaking, cautiously pour into the
solution a little concentrated sulphuric acid, a reddish-violet coloration
appears, which soon takes on a beautiful reddish violet shade. The
absorption spectrum shows a wide, faint band in the orange (A 615-670),
and a second, obscure, band in the green (A. 555-540).

F. G. Hopkins and Cole ' were the first to prepare tryptophane in a pure

' S. P. L. Sorensen: Compt. rend. trav. Lab. Carlsberg, 6, 137 (1905); Z. physiol.
Chem. 44, 448 (190.5).

' E. Fischer: Z. physiol. Chem. 35, 227 (1902).

' Commercial pepsin was used in these experiments. The possibility, therefore,
remains that other tissue ferments might have acted in conjunction with the pepsin,
for the reason that the commercial preparation is obtained by extracting the mucous
lining of the stomach.

* E. Fischer and E. Abderhalden: Z. physiol. Chem. 40, 215 (1903).

' Cf. E. Stadelmann: Z. Biol. 26, 491 (1890). R. Neumeister: ibid. 26, 324 (1890).
M. Nencki: Ber. 28, 560 (1895)

• E. Rohde: Z. physiol. Chem. 44, 101 (1905).

' F. G. Hopkins and S. W. Cole: J. Physiol. 27, 418 (1901); 29, 451 (1903).

ALBUMINS OR PKOTELNS 153

state. They permitted bacteria to act on tryptophane, and obtained indole,
skatole, skatole-carboxylic acid, and skatole-acetic acid. The bacillus of
anthrax, and Bacterium coli, in a strong anaerobic solution, produce skatole-
acetic acid, while putrefactive bacteria, on the other hand, give skatole-
carboxjdic acid in conjunction with indole and skatole. The constitution
of tryptophane has been recently established by the synthesis of Ellinger
and Flainand.' It is indole-a-aminopropionic acid.

H

C

HC C— C.CH.,.CH(NH.,). COOH

II I il ' '

HC C CH
\ ^ \ /
C N
H H

Tryptophane

The common form is dextrorotary both in alkaline and acid solutions.

It may be mentioned that tiyptophane is related to a peculiar metabolic
pmduct of the dog, namely, kynurenic acid, which is a y-hydroxy-^
quinolin-carboxylic acid of the following constitution: -

H
N=C
/ \

C C.COOH

/ ^ ^

HC C— C

II I OH

HC CH

C
H

Kynurenic acid

The transformation of tryptophane into this acid is not yet understood.
Pos.sibly .some idea of the process may be obtained from the fact that a
compound rich in oxygen which can be converted into quinolin is found
together with tryptophane. At pre.sent, however, we do not know
whether this ox y -tryptophane corresponds to a primary decomposition
product from protein, or whether it is formed secondarily from trypto-
phane.

To the group of heterocyclic Iniilding material of protein histidine also

» A. Ellinger and C. Flamand, Bor. 40, :i020 (1907).

' E. Abderhalden and M. Kempe; Z. physiol. Cliera. 52, 207 (1907).

154 LECTURE VIII.

belongs. We are indebted to A. Kossel ' for its discovery among the
cleavage-products of the protamine, sturine. It was for a long time
assigned to the di-amino acids, also called hexon bases. Pauly ^ has
recently succeeded in throwing some light on its constitution. He gives
it the formula of a-amino-^-imidazol-propionic acid:

CH— NH

II >H

C N"^

i
CH2

I

*CH . NH2

COOH

Hist id me

F. Knoop and A. Windaus,^ through further investigations, have shown
this constitution to be correct. The compound is la?vorotary and has an
alkaline reaction.

Only three di-amino acids are, as yet, known. These are lysine, arginine,
and di-amino-tri-hi/droxydodecoic acid. The constitution of the latter is not
yet determined. It was separated from casein by Emil Fischer and Emil
Abderhalden.'' There are many indications that it, or other closely allied
substances, occurs in other proteins.

Drechsel ^ first isolated lysine. He noticed in the decomposition of
casein by hydrochloric acid that other substances besides ammonia and
mono-amino acids were formed, which possessed a strong basic character.
Among these lysine was found. Drechsel concluded that it was probably
a di-amino-caproic acid. Ellinger * proved that this supposition was
correct. He allowed putrefactive bacteria to act on lysine, and after a
while obtained pentamethylenediamine (cadaverine). Ladenburg has
shown' that cadaverine has the following constitution:

CH2 . CH2 . CH2 . CH2 . CH2

I I

NH2 NH2

Cadaverine

' A. Kossel: Z. physiol. Chem. 22, 177 (1896-97); Sitzber. Akad. Wiss. Berlin, 1896.

» H. Pauly: Z. physiol. Chem. 42, 508 (1904).

' P. Knoop and A. Windaus: Hofmeister's Beitr. 7, 144 (1905); 8, 407 (1906).

* Fischer and Abderhalden: Z. physiol. Chem. 42, 540 (1904).

' E. Drechsel: Ber. 21, 117 (1889); Arch. Anat. Physiol. 1891, 254. E. Schulze and
E. Winterstein: Ergebnisse Physiol. (Asher and Spiro), 1, 32 (1902).

« A. Ellinger: Z. physiol. Chem. 29, 334 (1902). Cf . Ber. 31, 3183 (1899) ; 32, 3542
(1900).

' Ladenburg: ibid. 19, 780 (1886).

ALBUMINS OR PROTEINS. 155

We can assume that cadaverine is formed by splitting off carbon dioxide
from lysine, which has the empirical formula C6H14N2O2 :

*
CH2 . CH2 . CH., . CH2 . CH . COOH

I I

NH2 NH2

Lysine

= CHo . CHo . CHo . CH2 . CH2 + CO2

I " I

NH2 NH2

Cadaverine

By synthesizing the inactive lysine, Emil Fischer and Fritz Weigert ' have
shown that lysine is, as the above formula indicates, an a-, e-, diamino-
caproic acid.

E. Schulze - has also found lysine in germinating plants. It is widely
distributed among the proteins and is rarely absent from them. It reacts
strongly alkaline. As yet it has not been obtained in crystalline form.
It rotates polarized light towards the right.

Arginine was discovered by E. Schulze and Steiger ' in the cotyledons of
lupine seeds, and in the etiols of germinating pumpkin seeds. It rotates
towards the right, and likewise reacts alkaline. It was soon shown that
arginine could not be considered an individual compound in the same .sense
as the other amino acids mentioned. E. Schulze and E. Winterstein *
then showed that arginine yields urea and a base, on treatment with baryta
water. Schulze and Winterstein isolated this base by forming its benzoyl
derivative. This had the same composition and properties as a di-benzoyl
compoimd isolated by Jaffe' from the excreta of hens, which had been
fed benzoic acid. It proved to be a benzoyl compound of ornithine,
and was called by Jaffe, ornithuric acid. Jaffe recognized ornithine as
diaminovaleric acid. Ellinger" corroborated this view in the same manner
as was done with lysine, by acting on ornithine with putrefactive bacteria
and obtaining tetTamethijlenediamine (putrescine). By this conversion
it was shown that the two amino groups occupy the a and 8 positions.
The splitting of ornithine occurs in the following manner:

CH, . CH2 . CJIo - f'H . COOH = CII2 . CH2 . CH2 . CH2 + CO2
NH2 XH2 NH2 NH2

' E. Fischer and F. Weigert: Sitzber. Akad. Wiss. Berlin, 1902; Ber. 35, 3772 (1902).
' E. Schulze: Z. phy.siol. Chcm. 24, 18 (1S9S); 30, 270 (1900); 28, 465 (1899).
' E. Schulze and E. Steiger: ibid. 11, 4:? (1887); Ber. 30, 2879 (1898).

* E. Schulze and E. Winterstein : Z. physiol. Chera. 26, 1 (1898); Ber. 30, 2879 (1898).
'' M. Jaffe: ibid. 10, 1925 (1877); 11, 401 (1878).

• A. EUinger: loc. cit.

156 LECTURE VIII.

The position of the carboxyl group had not been determined. Emil
Fischer ' finally cleared up the constitution of ornithine by its synthesis.
By this it was definitely determined that ornithine was an a-, 3-, diamino-
valeric acid.

Thus one of the decomposition products of arginine, the ornithine, was
identified. It was next to be decided in what form the urea which was
obtained from arginine occurred in it. E. Schulze and E. Winterstein
suggested that a guanidine derivative was united to the ornithine. The
separation of urea and ornithine from such a compound would proceed in
the following manner:

NH2 NH2

I I

NH = C— NH— CH2— CH2— CH2— C^H— COOH + H2O =

Arginine

NH2 NH2 NH,

/ I I

C=0 + CH2— CHo— CHo— C'H— COOH
\ *

NH2

Urea Ornithine

Schulze and Winterstein finally succeeded in synthesizing arginine from
ornithine and cyanamide, thereby establishing the constitution of arginine.

NH2 NH2

I I

CHo— CH2— CH2— tlH— COOH + CN— NH2 =

Ornithine Cjianamide

NH2 NH2

NH = C— NH.CHo. CHo. CH2 . CH.COOH

Arginine

B. B^nech and F. Kutseher ' succeeded in obtaining guanidine from
arginine, by oxidation with barium permanganate.

We have only one group of the remaining protein decomposition
products to consider; that is, those containing sulphur. We have pre-
viously mentioned that sulphur is one of the essential constituents of
albumin. It is only absent from the protamines. The occurrence of
sulphur in proteins was recognized at an early date. It had been noted
that on boiling albuminous material with alkalies considerable amounts

' E. Fischer: Sitzber. Akad. Wiss. Berlin, 1900; Ber. 34, 454 (1901).
' E. Schulze and E. Winterstein: Z. physiol. Chem. 34, 128 (1901).
s E. B^n^ch and Fr. Kutseher: ibid. 32, 278 (1901).

ALBUMINS OR PROTEINS. 157

of alkaline sulphides were split off.' Fleitmann - was the first to observe
that onlj- a part of the sulphur was split off by the action of alkalies, while
another portion remained unacted upon. From these observations he
differentiated between oxidized and un-oxidized sulphur in albumin.
The latter, only, was susceptible of being split off. This distinction later
on caused much misundei-standing among investigators of the sulphur
components of albumin. A. Kriiger ' is entitled to much credit for having
shown the futility of this classification. He makes a distinction between
" loosely- " and " firmly- " combined sulphur. Fr. N. Schulz * proved
that such a distinction was more justifiable, by showing that one of the
sulphur cleavage-products of albumin, cystine, only gave off part of its
sulphur on boiling with alkali; in fact, little more than half. Various
albuminous substances, such as keratin (from the horn of cattle, and
human hair), serum-albumin, and serum-globulin, acted in the same
manner as cystine. Morner ^ then succeeded in obtaining large quantities
of cystine from the above-mentioned proteins, and showed that this amino
acid is probably the only sulphur compound present. Other proteins cer-
tainly contain other sulphur compounds besides cystine.

.\mong the decomposition products containing sulphur, cijstine is the
only one that has been positively identified. WoUaston," in 1810, first
isolated this from a renal calculus. Since then it has been separated
also from organs. Kiilz ' first isolated it from digestive solutions of fibrin,
and Emmerling * found it in horn. The wide distribution of cystine as a
decomposition product of albumin is now generally acknowledged.

The constitution of cystine has only recently been established; it is an
a-diamino-;3-dithio-dilactylic acid:

CH.— S— S— CH2

I I

*CH.(NHo) *CH(NH2)

I I

COOH COOH

Cystine

' The sulphur content of the proteins was at one time a factor of great importance
in the vie%vs prevailing conccrninK tlieir constitution. Cf. E. Friedmann: Ergeb.
Physiol, (.\sher and Spiro) 1, 15 (19012). E. Abderhalden: Biochera. Zentr. 2, 257
(1904).

' T. Fleitmann: .\nn. 61, 121 (1S47); 66, .380 (1848).

' A. Kruger: Pfluger's Arch. 43, 244 (1888).

' Fr. N. Schulz: Z. physiol. Chem. 25, 16 (1898).

' K. A. H. Morner: Md. 28, .V)r> (1899); 34, 207 (1901-02).

' WoUa-ston: Phil. Trans. 1810, 220.

' E. Kiilz: Z. Biol. 27, 415 (1890).

' O. Emmerling: Verh. Ges. Naturforsch Aerzte. 2, .391 (1894).

158 LECTURE VIII.

By reduction, we obtain cysteine from it, which is an a-amino-/3-thio-
propionic acid:

CHo . SH

I

CH. (NH^)

I
COOH

Cysteine

Cysteine, therefore, is closely related to alanine and serine. Friedmann '
oxidized cysteine and obtained cysteic acid:

CH2 - SO2 . OH

CHCNHs)

COOH

Cysteic acid

from which, by splitting off carbonic acid, we obtain taurine:

CH2 . SO2 . OH

I
CHaCNHz)

Taurine

This indicated an important relationship between a product related to
taurocholic acid and cystine. The correctness of the formula of cystine
as presented by E. Friedmann, and soon after by C. Neuberg,- has recently
been strengthened by the synthesis of cystine by Erlenmeyer.^

Observations by Baumann and Preusse * do not harmonize with the
above formula. They found that when brombenzene was fed to a dog,
the excreted urine contained a compound containing bromine, nitrogen,
and sulphur in its composition. Its composition was CnHioBrNSOa.
Baumann and Preusse designated this compound: Bromphenyl mer-
capturic acid. From, this, acetic acid and a compound, C9HioBrNS02,
are obtained by hydrolysis. The latter formula corresponds to the
empirical formula of cysteine, if the bromphenyl residue is replaced by
a hydrogen atom. The compound formed together with acetic acid was
therefore considered as bromphenyl cysteine. From the resulting cleavage-

' E. Friedmann: Hofmeister's Beitr. 3, 1 (1902).

' C. Neuberg: Ber. 35, 3161 (1902). Cf. K. A. H. Momer: Z. physiol. Chera. 42,
349 (1904).

' Erienmeyer: Jr. Ber. 36, 2720 (1903).

« E. Baumann and C. Preusse: ibid. 12, 806 (1879); Z. physiol. Chem. 5, 309 (1881).

ALBUMINS OR PROTEINS. 159

products, Baumann and Preusse proposed the following formula of
mercapturic acid:

CH3

I
CH3CO— NH . C— S . C6H4Br

I
COOH

If this formula be correct, then it must correspond to a differently
constituted cysteine than the above one. We might expect that cystine
could occur in various modifications. E. Friedmann,' proceeding from this
hypothesis, undertook to prove the constitution of mercapturic acid, and
showed that such an assumption was unnecessary, for he found that mer-
capturic acid and cysteine have their amino and thio groups analogously
situated :

CH-, . SH CHo . S . C6H4X

I ■ 1

CH . NH. CH . NH . CO . CH3

I ■ I

COOH COOH

Albumin-cysteine Mercapturic acid

Recent investigations have also shown that probably only one cysteine
exists.- Patten ^ has shown that only cystine and not cysteine occurs in the
original albumin molecule.

It is necessary to mention at this point that all the compounds so far
discussed, with the exception of phenylalanine, can also be obtained by
the hydrolytic action of ferments upon the albumins. Phenylalanine, as
already indicated, is found as such in plant seeds. As fermentation
hydrolysis undoubtedly furnishes the mildest possible form of decom-
position, we are justified in concluding that the cleavage-products of
protein which have been just mentioned exist as such in the albumin
molecule.

While discu.ssing the proteids, we mentioned the so-called " gluco-
proteids " of the albumins, which are characterized by a high percentage
of glucosamine, and, possibly, other carbohydrates. On account of the
firmness with which these groups are attached to the albumin molecule,
it is at present considered more correct to place them with the simpler
proteins rather than with the complex varieties. We can easily imagine
that glucosamine is held in combination in a manner analogous to that
which unites the amino acids to one another. We can also easily indicate

• E. Friedmann: Hofmeister's Beitr. 4, 486 (KOS).

' E. Fischer and U. Sii2uki: Z. physiol. Chem. 46, 405 (1905i.

' A. J. Patten: <bid. 39 350 (1903).

160 LECTURE VIII.

the close relationship of glucosamine to the amino acids as well as to the
carbohydrates. This is very evident from the following comparison:

CH2 . OH CH2 . OH CH2 . NH2

CH .OH CH . OH CH2

CH .OH CH . OH CH,

1 ' I

CH .OH CH . OH CH

CH .OH CH . NH, C

i

H. NH2

H : 0 CH : O COOH

Glucose Glucosamine Lysine

It is not right to consider as exceptional the albumins containing carbo-
hydrate groups. It is more correct, according to our present knowledge,
to speak of albuminous bodies characterized by a high content of gluco-
samine, just as we know of proteins which have considerable glycocoll.
Just as there are proteins which contain only small amounts of glycocoll,
and some with none at all, so we also recognize proteins which possess
small quantities of glucosamine, as well as those which have none of this
hexose base. The fact that other amino sugars probably participate in
the constitution of proteins does not affect this conception. It is doubtful
whether nitrogen-free sugars occur in albumin. It must also not be
forgotten that the presence of glucosamine, as a primary cleavage-product,
is doubted. A complex carbohydrate has been assumed to be the ante-
cedent of the glucosamine. The results at present are not sufficiently
exact to settle the question.' The conception that the " carbohydrate-
group " is a constituent of the albumin molecule in the same sense as the
amino acids, is rendered somewhat improbable by the fact that various
observers have obtained entirely different carbohydrate values in the
analysis of one and the same substance. It has even been suggested that
specific proteins, e.g., serum-albumin, unite with the sugars, conduct
them to the tissues, and finally give them up to the latter. Such an
assumption would be comprehensible if it were known that the carbo-
hydrate groups were loosely bound to the albumin molecule. This,
however, is not the case. A more satisfactory explanation would be
that the varying carbohydrate content of the proteins was due to the
albumins investigated not being identical.

As yet we know but little about the quantities of glucosamine present
in the mucins, — the proteins richest in carbohydrates. It is only known

' L. Langstein: Ergebnisse der Physiologie (Asher and Spiro), 1, 63 (1902); 3, 453
(1904).

ALBUMINS OR PROTEINS. . 161

that the mucins and their related substances can contain as much as 30 per
cent glucosamine. It is not at all unreasonable to expect varying results
from the same mucin, because it is absolutely impossible to purify these
compounds thoroughly. It is more striking that egg-albumin, which
is so easily crystallized, does not give concordant results on the amount
of glucosamine. It must also be remembered that egg-albumin contains,
besides albumin and globulin, ovi-mucoid, which contains about 30
per cent glucosamine. By the recent investigations of Fr. N. Schulz
and Zsigmondy,' it was shown how e.Ktremely difficult it is to free
egg-albumin from colloidal substances, even after sixfold crystalliz-
ation. In recrystallized egg-albumin, values varying from 16 to less than
one per cent of glucosamine were found.- We do not err, in assigning the
fluctuations of the carbohydrate content, which far exceed the analytical
error, to this cause of varying purity of the substance under investiga-
tion. It is, therefore, not yet determined whether egg-albumin is entitled
to a " carbohj'drate group." The same holds true regarding serum
albumin. This, also, invariably incloses some serum-mucoid,' which is
relatively rich in glucosamine. In fact, serum-globulin is distinctly
different, in that, aside from a trace of glucosamine, it also splits off
grape-sugar. Now serum-globulin is obtained by precipitation with
ammonium sulphate solution. To purify this, to the extent possible
with the crystallizable proteins, is entirely impossible. Besides grape-
sugar, serum also contains small quantities of other carbohydrates of
unknown constitution. It is very probable that the precipitated serum-
globulin contains such a carbohydrate mixed with it. Up to the present
time, we have had no investigation which would warrant us in assigning
any nitrogen-free carbohydrates to the albumin molecule.

If we sum up what we know about the " carbohydrate groups "
of the proteins, we will conclude that the mucins and mucoids contain
such groups; while although the remaining proteins may contain carbo-
hydrates, their presence has not been proved positively.

It is very important that absolute clearness should prevail in regard to
this question. We shall see later, that many facts make it seem probable
that carbohydrates are formed from albumin. The assumption that,
according to our present knowledge, the " carliohydrate group " of the

' Fr. X. Schulz and Zsigmondy: loc. cit.

' E. Abderhalden, P. Bergell, and T. Dorpinghaus: Z. physiol. Chcm. 41, 530 (1904).
Direct experiment .sliowed that repeated recrystallization reduced the quantity of
glucosamine present in albumin. Albumin crystallized once gave 7 per cent, three
times gave 4 per cent, while the seventh time showed only 2.5 per cent glucosamine.
That the values in this case are higher than in the work just mentioned, is due to the
fact that the crude osazone was weighed, while in the former the analytically pure
osazone was used as a basis for the calculation.

' C. N. Zanetti: Ann. Chim. Farmac. 12, 1897.

162 LECTURE VIII.

proteins is the source of the sugar formation from albumin, is without
justification, as we have just said. If sugars are formed from albumin,
then undoubtedly the amino acids are to be considered as their immediate
antecedents. It may be mentioned in addition that glucosamine espe-
cially is apparently not utilized at all by the organism for the production
of glycogen.

It is certainly not without significance that the mucins and mucoids,
proteins which are also widely distributed among the invertebrates,
should contain glucosamine — an amino-hexose — which is known to be
the basis for the formation of chitin.

The presence of carbohydrates in the albumins is indicated by certain
color reactions. If we add a few drops of an alcoholic solution of a-naphthol
to a solution of albumin, and allow a layer of concentrated sulphuric acid
to flow beneath this, a violet ring appears at the junction of the two
fluids. On shaking, the whole solution takes on a violet tinge. On
adding alcohol, ether, or caustic potash to this, the coloration becomes
yellow. If we use thymol instead of the a-naphthol, the coloration
produced is carmine-red. It turns green on dilution. These reactions —
called the " Molisch sugar tests " ' — depend on the formation of furfurol
from the carbohydrates present, by the action of sulphuric acid.

Another test which has been supposed to indicate the presence of a
carbohydrate group in proteins is the violet to deep blue color obtained
by boiling with fuming hydrochloric acid, when they have previously
been hydrolyzed. This is known as " Liebermann's reaction," but it is
not certain that this albumin reaction is due to carbohydrates.

In this connection we must call attention to two other albumin reactions.
If we add strong nitric acid to an aqueous solution of albumin, a yellow
coloration appears, very often in the cold, although generally only on
boiling. If we add an excess of caustic soda to this, the solution becomes
reddish brown; while if ammonia be used, an orange color results. This
is called the " xantho-proteic reaction," and depends on the formation of
nitro-derivatives, and, according to Salkowski,- requires the presence of
aromatic groups.

All the reactions which have now been mentioned for albumin, require
the presence of specific groups, and only apply to such proteins as contain
them. The blackening, which results when a protein is heated with
caustic alkali and a lead salt, is characteristic of a group containing sul-
phur. It depends on the formation of lead sulphide. Millon's reagent
indicates the tyrosine group; the glyoxylic acid characterizes the trypto-
phane combination. The carbohydrate group is detected by the Molisch
reaction, and also, possibly, by the Liebermann reaction. The xantho-

' Molisch: Monatsh. 7, 198 (1SS8).

2 E. Salkowski: Z. physiol. Chem. 12, 211 (1887).

ALBUMINS OR PROTEINS. 163

proteic reaction indicates the presence of aromatic groups. We are also
acquainted with another important color reaction, which does not properly
characterize any group as such. This is the so-called " Biuret-reaction."
If we freely add caustic soda or potash to an albumin solution, and
then carefully, drop by drop, a dilute solution of copper sulphate, a
blue to rose-violet coloration appears, which goes over into a blue on the
addition of more copper sulphate. The higher decomposition products
of albumin, the peptones, give a red coloration.

The cleavage-products of protein just mentioned, have been obtained
by the hydrolytic action of acids and of alkalies. We can easily imagine
that in these cases secondary decompositions take place. A number of
scientists doubted the occurrence of so many amino acids, and preferred
to assume that the proteins contained groups which gave rise to the forma-
tion of these various amino acids during the hydrolysis brought about by
the reagents.' It were conceivable that ornithine, proline, and amino-
valeric acid originate from the same atomic grouping; also lysine and
leucine, on the one hand, and tyrosine and phenylalanine on the other.
Such a conclusion does not harmonize with our present knowledge of the
actions of acids and alkalies, because they invariably yield the indi-
vidual amino acids in the same quantities. That these amino acids occur
in the proteins is very evident from their appearance, as such, in sprouting
plants, and even in the animal organism imder specific conditions. The
most important proof of their original occurrence is indicated by their
appearance during digestion. The albumins are broken down by the
hydrolytic action of ferments, especially by trypsin, into amino acids.
The decomposition by fermentation is the mildest imaginable. It takes
place at 37° C. .^11 the known amino acids have been obtained from diges-
tion mi.xtures except phenylalanine and diamino-trihydroxy-dodecylic
acid. The latter has never been looked for, while the former does not
appear in a state of combination which is accessible to the proteolytic
ferments.

The albumin as it reaches the digestive organs of an animal is subjected
to the action of two proteolytic ferments, pepsin and trypsin. Later on
we shall go more into detail regarding the behavior of the albumins during
the process of natural digestion. We shall, at present, devote our atten-
tion to the subject of artificial digestion, i.e., the digestion of the protein
outside of the alimentary tract. We must say that the results obtained
in these investigations do not harmonize in matters of detail. This is
mainly due to the different methods employed in using these ferments.
Until recently, the physiological chemist utilized extracts of orgarus whether
of the stomach or of the pancreatic gland, and the extirpated organs

' O. Loew: Hofmeister's Beitr. 1, 507 (1900).

164 LECTURE VIII.

themselves. We are, however, aware that many ferments occur in the
tissues, which are far more energetic in metaboUc processes, and act
in another direction, than the digestive ferments. Many of the
results obtained are undoubtedly due to the interaction of these tissue-
ferments. We are now able to obtain the digestive fluids in purest form,
thanks to the excellent methods originated by Pawlow ' and his students.
It is possible, on the one hand, to prepare a small special stomach, that is,
a pouch obtained by tying up a part of the walls of the stomach so as to
form a blind-sack, from which the pure gastric juice may be obtained with-
out the least admixture of any food residues. On the other hand, we can
obtain absolutely pure pancreatic juice, clear as water, by making a
pancreatic fistula, i.e., by grafting into the abdominal wall that part of the
mucous membrane of the duodenum at which the pancreatic duct enters.
As we shall see later, this fluid is inactive when the piece of intestinal
mucous membrane carrying the papilla is cut away. It must first be
made active preferably by the addition of intestinal juice. It is only
possible to obtain absolutely correct results by utilizing such ferment
solutions.

Amino acids do not immediately appear when the proteins — edestin,
for example — are undergoing digestion. We observe first of all that the
albumin is dissolved.- We notice at the same time that the digestion
mixture contains dialyzable substances which are not amino acids.
The digesting mixture may even be boiled without causing coagulation.
It has been held that the protein molecule by hydrolytic cleavage is
decomposed into products with lower molecular weights; the higher of
these are known as albnmoscs, from which in turn peptones are formed.
There is no sharp distinction between these two classes. Strictly speak-
ing, the conception of albumoses and peptones is not a chemical, but a
biological one, and we shall treat of them here as forming one class, and
drop the term "albumose." It represents, instead of certain chemical
individuals, a group of compounds which exist temporarily in a similar
condition. For the present, these names do not signify much to us.
Not content with this distinction of the two groups of substances,
scientists have classified them according to their solubiHty relations, —
according to the extent to which they may be precipitated, etc., — thereby
designating them with new names. It has also been found that the
peptones obtained from different proteins are not identical, so that they
have been named according to the protein from which they are formed.

' J. P. Pawlow: Ergebnisse d. Physiol. (Asher and Spiro) 1, 246 (1902).

' The earliest observations on tryptic digestion were made by Corvisart: Gaz. Heb-
dom. Nos. 15, 16, 19 0857). W. Kiihne: Virchow's Arch. 39, 130 (1867). Cf. also
E. Abderhalden: Z. physiol. Chem. 44, 17 (1905).

ALBUMINS OR PROTEINS. 165

Thus we speak of globuloses, vitelloses, etc. Undoubtedly, we shall even-
tually find that a great deal of this difference in behavior is due to the
different amino acids, which are contained in the different proteins, and
their arrangement in the molecule, so that before long we shall be able to
replace this purely biological conception by a chemical one. For the present
the investigations have gone beyond our actual knowledge, and have led to
certain results, which do not yet rest upon a firm foundation. For this
reason we shall not attempt to describe any of the numerous special albu-
moses and peptones, but simply content ourselves with the conception
itself. The albumoses are in general characterized by the fact that they
are precipitated when their solutions are saturated with ammonium sul-
phate, while the peptones then remain in solution. By means of the
behavior of a digesting mixture towards ammonium sulphate, we can
determine how far the digestion has already gone.'

Up to this point the changes produced upon the protein molecule by
the pepsin-hydrochloric acid of the stomach and the trypsin of the pan-
creas are apparently quite similar. In both cases albumoses and pep-
tones are formed. Of course the action of pepsin may nevertheless be
entirely different from that of trypsin in spite of this external similarity.
It may be that a different place in the protein molecule is attacked.
Unquestionably even' in gastric digestion a large quantity of products
are obtained which represent lower products than the peptones, and
some of these do not even give the biuret reaction. Simple amino acids,
however, with the exception of traces of tyrosine, have not been found
here.'

Tryptic digestion goes much farther. We quickly observe crystalline
depositions on the walls of the vessel in which the digestive mixture is
placed. This is tyrosine, which separates on account of its difficult solu-
bility. It is very quickly split off from the albumin molecule. In 48
hours, and even in less time, the entire tyrosine content of the albumin can
be isolated as such.' In the digestion of edestin from cotton-seeds, for
example, the following observations were made: *

PERCENT.VGE OF TYROSINE OF THE TOT.VL AMOUNT OCCURRING
IN EDESTIN.

Time of digestion

' See page 188.

• Emil .\bderhalden and Otto Rostoski: Z. physiol. Chem. 44, 265 (1905).
' E. .■Vbderhaldcn and B. Reinbold: Z. physiol. Chem. 44, 284 (1905).

• Ibid. 46, 159 (1905).

166

LECTURE VIII.

Tryptophane, as well as cystine, can be obtained just as quickly as
tyrosine; the former being easily recognized by the violet color which is
formed when bromine water and acetic acid are added to the digesting
liquid. The remaining amino acids are obtained later on. This has been
successfully proved in the case of glutamic acid. The following per-
centages of the total amount of this amino acid occurring in edestin, were
obtained:

Time of digestion 1 day.

4.3

2 days.
7.4

3 days.
10.9

8 days.
31. i

16 days.
60.2

Alanine, leucine, amino-valeric acid, and aspartic acid, acted in the
same manner; while a-proline and phenylalanine could, in no case, be
separated from a digesting fluid.

The following observations have given us an explanation of this peculiar
behavior: ' If we digest casein, edestin, serum-globuhn, egg-albumin,
hemoglobin, or fibrin with pancreatin,^ or even with pancreatic juice, we
obtain all the mono-amino and di-amino acids in the digesting mixture,
with the exception of proline and phenylalanine. These amino acids do
not occur, or, if so, only in minute quantities, even when tryptic digestion
precedes that of the pepsin-hydrochloric acid.' Now we can precipitate
even from a greatly diluted digesting mixture, by means of phospho-
tungstic acid, a product which apparently is a mixture of highly compli-
cated compounds. It sometimes gives the biuret reaction; then again,
no result is obtained — according to the time of digestion. No free amino
acids can be isolated from this product, although we can obtain such by
hydrolysis with fuming hydrochloric or 25 per cent sulphuric acid. In the
presence of small amounts of alanine, leucine, aspartic acid, and glutamic
acid, we obtain large amounts of a-proline and phenylalanine; and, in
those proteins containing glycocoll, even this amino acitl, in amounts
approximating those contained in the protein in question. The proteins
evidently contain groups which resist the action of ferments. Of
especial interest is the fact that here also the rate at which the individual
amino acids are separated varies.

From these investigations it is clear that fermentative decomposition is a
progressive one. An immediate disruption of the protein does not occur.

' E. Fischer and E. Abderhalden: Z. physiol. Chem. 39, 81 (1903).

' A commercial preparation of pepsin was used in this experiment, consequently
impure. It is very probable that the latter disintegrated albumin more than the gastric
juice alone would do.

' E. Fischer and E. Abderhalden: Z. physiol. Chem. 40, 215 (1903).

ALBUMINS OR PROTEINS.

167

The following diagram will give an idea of the hydrolysis brought about
by means of the pancreatic ferment, trypsin:

Protein
I

i
/Peptonesv

A mixture of complicated compounds
composed of various amino acids.
" Polypeptides."

Tyrosine, tryptophane, cystine, alan-
ine, aminovaleric acid, leucine,
aspartic acid, glutamic acid, histi-
dine, lysine, and arginine.

The product consisting of amino acids still combined with one another,
and which may for the present be designated as " polypeptides," is dif-
ferent in the case of different proteins. In the case of edestin the amount
is smaller than in the case of casein, and that obtained from the latter
is less than from serum-globulin.

From the investigations of Abderhalden and Reinbold,' it has been
clearly shown that, even the peptones, which still give the characteristic
red biuret reaction, do not immediately break down into amino acids.
There are certain!}* intermediate products between the peptones and the
amino acids. Here, again, the progressive decomposition is clearly
evident. Doubtless the simpler peptides, which we will shortly discuss,
also appear as intermediate products. We shall return to this shortly.

The most important result obtained from these investigations is, that
the amino acids, which split off from albumin by the action of alkalies and
acids, are already formed in the albumin molecule and are not formed by
a secondarj^ process; and, further, that in spite of the early appearance of
.crystalline cleavage-products, the fermentative decomposition need not
necessarily be far advanced. All the tyrosine occurring in a digesting
mi.xture can be detected, even if, for instance, only seven per cent of the
amount of glutamic acid occurring in the albumin has been set free.

Besides pepsin and trypsin, we have to consider erepsin, which occurs in
the alimentary tract as an albumin-splitting ferment, and has been isolated
by 0. Cohnheim.^ It does not act on the proteins themselves, but only
on their decomposition products, the peptones. The only exceptions
to this rule are casein, protamines, and histons; these are attacked

' Loc. cit.

' O. Cohnheim: Z. physiol. Chem. 33, 451 (1901); 35, 134 (1902).
Salaskin: ibid. 35, 419 (1902).

Cf. also S.

168 LECTURE VIII.

by erepsin. The decomposition products are the same as those produced
by trypsin. At present it is difficult to pass judgment on the actual
existence of this ferment. According to the investigations of Vernon,'
it occurs widely distributed in the animal kingdom, and is to be found in
all tissues. It is very difficult to decide whether the proteolytic ferments
should be considered as homogeneous or as mixtures of ferments of differ-
ent individual functions. It is not unreasonable ^ to assume that for each
protein, or for a class of these substances, a special series of ferments exists.
On the other hand, we could easily imagine that one ferment follows
another, step by step, in the decomposition, in the same manner as is
true with the carbohydrates, in which case diastase decomposes them
only to the maltose stage, leaving the latter to be further acted upon by
maltase.'

As a matter of course, proteolytic ferments must also be active in the
tissues and cells, and many observations indicate that the action is anal-
ogous to that of trypsin. This applies not only to the animal cells, but
also to those of plants. Especially noteworthy is the ferment papayotin
occurring in the milk of the melon, Carica papaya. It quickly dissolves
albumin. Its action appears to be similar to that of trypsin.'' Other
active ferments have also been isolated from various plants, as, for instance,
from the sap of the fig-tree, Ficus carica, and macrocarpa. Other plants,
like the banana, are credited with possessing a ferment analogous to
pepsin.

Especial interest attaches to those plants which also secrete ferments
externally which correspond to the digestive fluids characteristic of the
animal organism. They constitute the large group of carnivorous plants.
We will mention merely the Drosera and Pinguicula, growing in peat
bogs; and the varieties of Vtricularia inhabiting brooks and stagnant
pools. The Nepente species, Dioncea muscipula, act on a larger scale. It
is still a question whether the action of the ferment produced is analogous
to that of pepsin or to that of trypsin. It has even been suggested that the
fermentative action of Nepente is due to bacteria.

In the cryptogams the proteolytic ferments are also widely distributed,
and in many cases have been detected.

Before discussing the manner in which the fundamental constituents of
the albumins are combined, we must devote a little attention to several
other substances which occur among the cleavage-products of the proteins,

' H. M. Vemon: J. Physiol. 32, 33 (1904); 33, 81 (1905).

' Cf. Lecture on Ferments.

' Cf. W. M. Bayliss and E. H. Starling; J. Physiol. 29, 174 (1903). K. Mays: Z.
physiol. Chem. 38, 428 (1903). L. Pollak: Hofmeister's Beitr. 6,95 (1904). K. Kiesel:
Pfliiger's Arch. 108, 334 (1905).

• O. Emmerling: Her. 36, 695 (1902).

ALBUMINS OR PROTEINS. 169

but which, nevertheless, probably do not occur as a constituent of the
original molecule.

Amongst these is leucinimidc. It is an anhydriile of leucine, and is 3-6,
di-isobutyl, 2-5, di-acipiperazine:

C4H9 . CH . NH . CO

I I

CO . NH . CH . C4H9

Ritthausen ' first observed this in an acid hydrolysis. Cohn ^ also
described it. Salaskin and Kowalewsky ^ recently even separated it,
although only in minute quantity, from peptic and tryptic digestion.* It
has not yet been decided whether Icucinimide occurs as such in the
albumin molecule. It is possible that it is formed by a secondary process,
perhaps from a leucyl-leucine.

Pyroracemic acid, CH3 . CO . COOK, discovered by Morner,^ is un-
questionably formed by a secondary reaction. It is probably produced
from alanine, serine, or cystine. The origin of a-thiolactic acid, discovered
by Suter," is problematical. It may possibly be derived from cystine,
although this has the thio group in the /? position. Ornithine, which is
certainly a secondary decomposition product, is derived from arginine.

The albumins quickly undergo putrefaction.' They are also decomposed
by bacteria in the intestines. It is necessary to become acquainted with
the compounds formed in this manner. They are all related to the amino
acids already mentioned. The bacteria decompose the albumin in the
same manner as do the proteolytic ferments, especially trypsin. Peptones,
and finally amino acids, are produced.

' Ritthausen: Die Eiweisskorper der Getreidearten, Bonn, 1872.

» R. Cohn: Z. physiol. Chem. 22, 153 (1896-97); 29, 283 (1900).

' S. Salaskin and K. Kowalewsk-y: Ontl. 38, 567 (1903).

* The author has him.self also tried to isolate leucinimide from peptic and tryptic
digestion products, but in vain. Something went into solution in the acetic ether. Its
easy solubiUty in dilute hydrochloric acid showed that it was not leucinimide. He,
however, succeeded in obtaining about one per cent of leucinimide by hydrolyzing
casein with 25 per cent sulphuric acid.

' K. A. H. M.irner: Z. phy.siol. Chem. 42, 121 (1901).

' Suter: ibid. 20, ,504 and 577 (18951; Ilofmoister's Beitr. 3, 184 (1902). K. A. H.
Morner: Z. physiol. Chem. 42, 365 (1901).

' E. and H. Salkowski: Z. physiol. Chem. 8, 417 (1884); Und. 9, 8 (1884); 9, 491
(1885); 27, 302 (1899). X. Nencki: Ber. 7, 1593 (1874); 8, 336 (1875); 10, 10.32 (1877);
J. pr. Chem. 26, 47 (1882); Z. physiol. Chem. 4, 371 (1880); Z. med. Wiss. 1878, 47.
Nencki: Opera omnia, vol. i, pp. 92, 113, 144, 244, 246, 674, 537, 354, 418, etc.
E. Baumann: Ber. 12, 14.50 (1879); Z. physiol. Chem. 4, 304 (1880); 6, 183 (1882); 7,
282 and 553 (1,895). E. Baumann and L. Brieger: ihuL 3, 149 .and 284 (1879). L.
Brieger: J. pr. Oicm. 17, 124 (1877); Ber. 10, 1027 (1S77); 12, 1986 (1879); Z.
physiol. Chem. 2, 241 (1878); 3, 1.34 (1879); 4, 414 (1880); 5, 366 (1881). Cf. also L.
Brieger: Die Ptomaine, Berlin, 1886.

170 LECTURE VIII.

The decomposition, however, does not end here. The bacteria break
these down in two directions. On one hand they split off the amino
acids. Simple acids remain: acetic acid is produced from glycocoll; pro-
pionic acid from alanine; valeric acid from amino-valeric acid; etc. The
5-amino- valeric acid, found in putrefying mixtures, may be produced
from ornithine, or by splitting off the pyrrole ring from a-pyrrolidine-
carboxylic acid. Tartaric acid, phenyl-propionic acid, p-hydroxyphenyl-
propionic acid, and skatole-acetic acid are also found. On the other hand,
carbon dioxide is split off from the amino acids by bacterial action. This
proc&ss gives us pentamethylenediamine (cadaverine) from lysine; while
tetramethylenediamine (putrescine) results from arginine and ornithine.

From phenylalanine we obtain phenylethylamine CeHs . CH2 . CH2NH2,
with evolution of carbon dioxide; and from tyrosine, oxyphenylethyl-
amine. As a rule the decomposition does not stop at these products.
They are oxidized. We can indicate the further destruction of tyrosine
(p-hydroxyphenyl-amino-propionic acid) as follows:

p-hydrox3rphenyl-amino-propionic acid C6H4 . OH. CH2. CH.NH2.COOH

7)-hydroxyphenyI-propionic acid C6H4 . OH . CH2. CH2.COOH

7)-hydroxyphenyl-acetic acid C6H4 . OH . CH2. COOH

p-hydroxymandelic acid C6H4 . OH . CH(OH) . COOH

p-cresol C6H4 . OH . CH3

phenol . CeHs . OH

In an analogous manner phenjdalanine (phenyl-amino-propionic acid)
breaks down through phenyl-propionic acid and phenyl-acetic acid.
Tryptophane (skatole-amino-acetic acid) gives skatole-acetic acid, skatole-
carbonic acid, skatole and indole. We shall meet with phenol, skatole,
and indole in the animal organism. They are produced in intestinal
putrefaction, and appear in the urine combined with sulphuric acid.
Sulphureted hydrogen is liberated from cystine.

LECTURE IX.

ALBUMINS OR PROTEINS.
III.
Composition of Individu.^l Proteins. Constitution.

In the formation of proteins, the amino acids alone participate, as far
as we know, with the single exception of the amino-hexose, glucosamine,
which occurs in many varieties of albumin. The number of these amino
acids already discovered is very large. It includes the following: glycocoU,
alanine, amino-valeric acid, leucine, isoleucine, a-pyrrolidine-carbo.xylic
acid (proline), oxypyrrolidine-carboxylic acid (oxyproline), serine, phenyl-
alanine, glutamic acid, aspartic acid, tyrosine, cystine, tryptophane, lysine,
histidine, arginine, and diaminotrihydroxydodecoic acid. It is of chief
interest to learn whether the proteins, at present known, contain the same
fundamental substances, or whether specific groups of proteins are char-
acterized by their content of individual amino acids. Another matter of
considerable importance is the relative quantity of the different amino
acids, occurring in the proteins. It is possible that the differences between
the various proteins are due to varying relations of the quantities of
individual amino acids present. On the other hand, it is a matter of the
greatest importance to know the quantitative amounts of these con-
stituents of the albumins for use in further work on this subject. We
should like to know how great a portion of the whole albumin molecule
is already understood. Unfortunately, we have no quantitative method
for estimating the amino acids. True, we can estimate very exactly some
of the cleavage-products, like tyrosine and glutamic acid, but for the
remainder of the amino acids we can only estimate the approximate
amounts. Our knowledge concerning protein formation was, until re-
cently, very limited.

Although various amino acids had been isolated, and the quantitative
relations of lysine, arginine, and histidine in different albumin molecules
had been established, through the researches of A. Kossel, investigators as
a rule attempted only to prepare the proteins in as pure a form as possible,
and to classify them according to their elementary composition. A turn-
ing-point in the whole chemistry of the albumins was reached when E.
Fischer ' introduced a new method for isolating the amino acids. Briefly,
the process consists in forming the esters of the mono-amino acids, and

E. Fischer: Z. physiol. Chem. 33, l.il (1901).
171

172

LECTURE IX.

separating them by fractional distillation. The amino acids are then re-
covered by saponifying the amino acid esters. On account of considerable
differences in the boiling-points of these esters, it is possible to obtain by
mere distillation a fairly satisfactory complete separation of the amino
acids. With the help of this method a considerable number of protein
substances have been carefully examined. We will give in the follow-
ing summary the results thus arranged according to the classification
previously given. It may be said that the amounts of amino acids
indicated, represent the minimum values. As the various proteins were
all analyzed under the same conditions, it is, therefore, possible to com-
pare the individual proteins according to their percentages of mono-
amine acids. The values given are all based on 100 grams of ash-free
material, dried at 100 degrees.

1. THE ALBUMIN GROUP.

Serum-
albumin.'

Egg-
albumin.'

0.
2.7
20 0
1.0
3.1
7,7
3 1
2.3
0.6
2 1
present

0

8 1

7 1

2 25

4 4

8 0

1 5

0 2

1 1

Tryptophane

present

E. Abderhalden: ibid. 37, 495 (1903).

E. Abderhalden and F. Pregyl: ibid. 46, 24 (1905).

2. THE GLOBULIN GROUP.

GlycocoU

Alanine

Aminovaleric acid

Leucine

« -Proline

Phenylalanine . ,
Glutamic acid . ,
Aspartic acid . . ,

Cystine

Serine

Tyrosine

Tryptophane . . ,
Oxyproline . . . ,

Lysine

Arginine

Histidine

Serum-
globulin.'

375
2.2
present
18.7
2.8
3.8
8,5'
2.5
0.7

2.5
present

Edestin

from

hemp-seed.^

3.8

3.6

present

20.9

1.7

2.4

6.3

4.5

0.25

0.33

2.1
present

2.0

1,0

1.7
11.1

Edestin

from

cotton-seed.

1.2
4.5
present
15.5
2.3
3 9
17.2
2.0

0.4

2,3

present

Edestin from

sunflower

seed .'

2.5
4.5
0.6

12.9
2.8
4 0

13.0
3.2

'0,'2

2.0

present

' E. Abderhalden: Z. physiol. Chem. 44, 17 (1905).
' E. Abderhalden and F. Samuely: ibid. 46, 193 (1905).
^ E. Abderhalden: ibid. 37, 499 (1903); 40, 249 (1903).
* E. Abderhalden and O. Rostoski: ibid. 44, 2G5 (1905).
» E. Abderhalden and B. Reinhold: ibid. 44, 284 (1905).

ALBUMINS OR PROTEINS. 173

3. GROUP OF THE PLANT-CASEINS, PHYTOVITELLINS, LEGUMINS, ETC.

Proteins

soluble

in alcohol.

Conglutin

Albumin

from

Gliadin '

Lupinus seeds.*

Pine seeds.*

from

Zein.'

wheat flour.

not

Glycocoll . . . .

0.9

determined

0.8

1.0

0.6

Alanine

2.7

0.5

2.5

2.8

1.8

Aminovaleric acid

0.33

present

1.1

1.0

present

Leucine

6.0

11.2

6 75

8.2

6.2

a-Proline ....

2.4

1.5

2.6

2.3

2.8

Phenylalanine . .

2.6

7.0

3.1

2.0

1.2

Glutamic acid . .

37.5

11.8

19.5

16.3

7.8

Aspartic acid . .

1.3

1.0

3.0

4.0

1.8

Serine

0.12

present

present

Tyrosine

2.4

10.1'

2.1

2.8

1.7

Tryptophane . . .

about 1.0

present

present

Lysine

0. 1

0. ']

2.1 1

5.5 1

0.25 1

Arginine

3.4 \<

1.82 «

6.6 «

4.6 »

10.9 «

Histidine . . . .

1.7

0.81 ]

0.65 J

1.1 J

0.62 J

' E. Abderhalden and F. Samuely: Z. physiol. Chem. 44, 276 (1905)

' L. Langstein: ibid. 37, 508 (1903).

» F. Kutscher: ibid. 38, 111 (1903).

' A. Kossel and F. Kutscher: ibid. 35, 165 (1900).

' E. Abderhalden and J. B. Herrick: ibid. 45, 479 (1905).

« E. Schulze and E. Winterstcin: ibid. 33, 547 (1901).

' E. Abderhalden and B. Babkin: ibid. 47 (1906).

" Abderhalden and Teruiichi: ibid. 45, 473 (1905).

4. GROUP OF FIBRINOGENS AND FIBRINS.'

Glycocoll • 3.0

Alanine 3.6

Aminovaleric acid 1.0

Leucine 15.0

a-Proline 3.6

Phenylalanine 2.5

Glutamic acid " 10.4

Aspartic acid 2.0

Serine 0.8

Tyrosine 3.5

Tryptophane present

' Abderhalden and Voitinovici :
Bruner : Diss. Berlin, 1905.

Z, physiol. Chem. 52, 308 (1907). Cf. also A.

174

LECTURE IX.
5. GROUP OF NUCLEO-ALBUMINS.

Casein

Casein

from

from

cow's milk.'

goat's milk.'

0.

0.

0.9

1.5

1.0

10.5

7.4

3.1

4.6

3.2

2.75

11.0

12.0

1.2

1.2

0 065 '

0 23»

4.5

4.95

1.5

present

0.75«

present

0.25'

5.80

4.84 '

2.59

Glycocoll

Alanine

Aminovaleric acid

Leucine

a-Proline

Phenylalanine

Glutamic acid

Aspartic acid

Cystine

Serine

Tyrosine

Tryptophane

Diaminotrihydroxydodecoic acid

Hydroxyproline

Lysine

Arginine

Histidine

Cf. also E. Abderhalden; loc. cit. and E. Fischer: Z. physiol. Chem. 33, 151 (1901).

K. A. H. Momer: ibid. 34, 207 (1901-02).

E. Fischer; ibiii. 39, 155 (1903).

E. Fischer and E. Abderhalden: ibid. 42, 540 (1904).

E. Hart: ibid. 23, 347 (1901).

E. Abderhalden and A. Schittenhelm : ibid. 47, 1906.

6. GROUP OF THE HISTONS.

Globin
from Oxy-
hemoglobin of
the horse.'

Glycocoll . . .
Alanine . . . .
Leucine . . . .
a-Proline . . .
Phenylalanine
Glutamic acid
Aspartic acid
Cystine . . . .

Serine

Tryptophane . .
Tyrosine . . .
Hydroxyproline

Lysine

Arginine . . . .
Histidine. . . .

' E. Abderhalden and P. Rona: Z. physiol. Chem. 41, 278 (1904).
» E. Abderhalden: ibid. 37, 484 (1903). Cf. also E. Fischer and E. Abderhalden:
ibid. 36, 268 (1902).

ALBUMINS OR PROTEINS.

175

7. GROUP OF THE PROTAMINES.

The protamines, as we have already mentioned, have been very carefully
studied by A. Kossel and his students. They are mainly composed of
di-amino acids. It is only recently that mono-amino acids have been
detected in the protamines. A. Kossel ' states that the protamines con-
tain only small quantities of specific mono-amino acids. An observa-
tion somewhat at variance with this, was made' in a very carefully
purified sample of salmine, but this may be explained perhaps by an
immature condition of the testes from which the preparation was obtained.
We have already mentioned that as a matter of fact the amounts of
di-amino acids isolated from the proteins in the testes of the salmon
depend on the maturity of the latter. It is very probable that the
protamines, which evidently can be traced back to the proteins of
the muscles, are formed from histons, which are to be considered as the
transition stage in the transformation from the proteins of the muscles
to the protamines. The salmine investigated contained alanine, leucine,
and a-proline, while phenylalanine and aspartic acid were also, undoubt-
edly, present.

Kossel and his students give the following amino acids as the constituents
of the protamines:

In 100 grams of Albumin are present

Arginine. Lysine. Histidine. Alanine

Salmine
Clupeine .
Cyclopterine
Scombrine
Sturine
Cyprinine .
Cyprinine II

gms.
87.4
82.2
62.5

+
58.2

4.9

+

gms.

0.

0.

0.

0.
12.0

gms.
0.
0.

6

12 9
0.
0.

Salmine . .
Clupeine . .
Cyclopterine
Scombrine
Sturine . . .
Cyprinine . .
Cyprinine II

In 100 grams of Albumin are present

Amino-
valeric
acid.

4.3 gms,

+

11.0 gms.

8.0 gms.

Trypto-
pbane.

' Cf. A. Kossel: Z. physiol. Chem. 40, 311 (1903). A. Kossel and H. D. Dakin: ibid.
40, 565 (1004); 41, 407 (1904).

' E. Abderhalden: ibid. 41, 55 (1904).

176

LECTURE IX.

8. GROUP OF ALBUMINOIDS,

GlycocoU ....

Alanine

Aminovaleric acid
a-Proline ....

Leucine

Phenylalanine
Glutamic acid . .
Aspartic acid . .

Cystine

Serine

Tyrosine ....
Lysine

Arginine ....
Histidine ....

Silk
fibroin.'

36.0

21.0

0.

present '

1.5

1.5

0.

present

i.e''

10.5
in small
amount;

1.0
in small
amounts

25.75

6.6

1.0

1.7
21.4

3.9

0.8
probably
present

0.34'
0.3«

horn.'

0.34
1.2
5.7
3.6
18.3
3.0
3.0
2.5

Keratin
from horse-
hair."

0.0
3.7
0.3

very much ' over 10 per
cent "

0.7
4.6

0.6
3.2

Keratin
from
goose-
feathers.*"

2.6
1.8
0.5
3.5
8.0
0.0
2.3
1.1

0.4
3.6

GlycocoU

Alanine

Aminovaleric acid

Leucine

a-Proline

Phenylalanine

Glutamic acid

Aspartic acid

Cystine

Serine

Tyrosine

Tryptophane

Lysine

Arginine

Histidine

Hydroxyproline

' E. Fischer and A. Skita: Z. physiol. Chem. 33, 177 (1901).

' E. Fischer: ibid. 39, 155 (1903).

5 Fischer and Skita; ibid. 35, 221 (1902).

« E. Abderhalden and A. Schittenlielm : ibid. 41, 293 (1904).

' H. Schwarz: *iV/. 18, 487 (1894).

' A. Kossel and F. Kutscher: ibid. 25, 551 (1898).

' E. Fischer and T. Dorpinghaus: ibid. 36, 462 (1902).

« K. A. H. Morner: 28, 595 (1899); 34, 207 (1901-02).

' E. Abderhalden and H. G. Wells: ibid. 46, 31 (1905).

"> E. Abderhalden and E. R. LeCount: ibid. 46, 40 (1905).

" E. Fischer, P. A. Levene, and R. H. Aders: ibid. 35, 70 (1902).

" E. Fischer: ibid. 35, 221 (1902).

" E. Fischer and E. Abderhalden: Z. physiol. Chem. 42, 540 (1904).

" E. Hart; ibid. 23, 347 (1901).

" E. Fischer: ibid. 35, 221 (1902).

ALBUMINS OR PROTEINS. 177

A glance at the composition of the different kinds of proteins shows that
they all, with the exception of the protamines, contain the same funda-
mental constituents. Occasionally one or another amino acid is absent;
thus, glycocoll does not occur in egg- or serum-albumin; lysine is absent
in the plant albumins which are soluble in alcohol; tyrosine and trypto-
phane in gelatin. If we compare the relative amounts of the various
amino acids occurring in the individual proteins, we notice appreciable
differences. This is especially striking if we compare the individual
groups. In the first place we notice the varying proportions of the
mono- and di-amino acids that go to make up the individual proteins.
The latter are very strongly represented in the protamines, and least so
among the albuminoids. Between these two extremes we find the com-
mon albumins and the histons. Very noticeable is the predominance of
special mono-amino acids, e.g., glutamic acid and leucine in the albumins
present in the seeds of plants. It constitutes one-third of gliadin.
If we compare the individual groups of proteins among themselves, we
find in many cases a very general similarity. Thus, glycocoll is absent
in egg-albumin and serum-albumin, whereas the globulins invariably con-
tain it. We are, therefore, in a position to classify chemically at least a
portion of the different proteins. The fact that they all contain the same
fundamental constituents makes it easier for us to understand their trans-
formations in the animal organism.

Although the complete hydrolysis of itself serves to give us a fairly
comprehensive knowledge of the amino acids participating in the con-
struction of the albumins, it, on the other hand, does not give us any idea
of the manner in which these substances are united. Until recently it had
not been possible to split off complexes from the proteins, and to identify
positively individual compounds containing only a part of the total amino
acids. We are certainly justified in concluding that the albumoses have a
lower molecular weight than the original proteins, and that the peptones
are, without doubt, to be considered as still lower cleavage-products. Up
to the present time the albumoses and peptones have been grouped almost
entirely according to their limits of precipitation and solubility. Only in
specific cases have they been characterized Ijy the absence of a definite
amino acid, or by an excess of such. Recently M. Siegfried and his
students have tried to obtain products by cautious hydrolysis of some
of the albuminous substances which would contain only a part of the
amino acids occurring in the original protein. Siegfried ' has described
several such products. He calls them kyrines. It is at present impossible

' Siegfried; Ber. math-physikal. Kl. klg. sachsischen Gesel. Wiss. Leipzig, Sitzung II.
III. p. 63, 1903; Z. physiol. Chem. 38, 259 (1903); 43, 46 (1904); 43, 44 (1904). Cf.
also C. Bockel: ibid. 38, 289 (1903). T. R. Kruger: ibid. 38, 320 (1903). W. Scheer-
messer: ibid. 41, 68 (1904). Z. H. Skraup and R. Zwerger: Monatsh. 26, 1403 (1905).

178 LECTURE IX.

to say whether they are individual substances or mixtures. Thus far their
study has not helped our knowledge regarding the construction of the albu-
min molecule. There is no doubt that the product obtained by Emil
Fischer and Emil Abderhalden by means of tryptic digestion, which did
not give the biuret reaction and was designated as " polypeptide," repre-
sents a cleavage product of a lower order of magnitude than the peptones.
It is very probably a mixture of various decomposition products. The
reason why we have not yet succeeded in getting an idea of the structure
of albumin by means of partial decomposition, is due to the fact that with
the large number of amino acids we should necessarily expect to find a
great many different decomposition products. For instance, in a digest-
ing mixture we find, besides peptones and free amino acids, other cleavage-
products which do not give the biuret reaction. As it is almost impossible
to separate the amino acids already known from such a mixture, it is, there-
fore, natural to e.xpect, considering our unfamiliarity with the higher com-
plexes, that we can hardly hope to isolate them in a satisfactory manner.

Recognizing this fact, Emil Fischer ' recently began to investigate the
constitution of the albumins from an entirely different standpoint. He
chose the synthetic method. By linking the amino acids together, com-
pounds must necessarily result which bear some relation to the albumins.
After obtaining a knowledge of the characteristics of these synthetic sub-
stances, it should be possible to devise ways and means to produce analo-
gous compounds from the albumins. We may say, at the start, that Emil
Fischer's early expectations have already been partially realized. While
the constitution of albumin was, until recently, very much in darkness, we
can now thank Emil Fischer and his students for their extensive researches
toward solving this problem. Emil Fischer's work will, undoubtedly,
constitute the foundations of both the chemistry and the biology of
the albumins. We must return to it in all phases of the question, and
shall, therefore, only briefly outline its fundamental characteristics here.

Emil Fischer started with the assumption that the amino acids in the
albumins were combined in the form of an amide-linking. He has shown
that the amino acids possess the ability of easily combining among them-
selves, thereby splitting off water, the amino group of one amino acid
reacting with the carboxyl group of another. The simplest representative
of this class of compounds, glycyl-glycine, is produced in the following
manner:

NH2 . CH2 . COOH -f HNH . CH2 . COOH — H2O

Glycocoll Glycocoll

= NH2 . CH2 ■ CO . NH . CH2 . COOH

Glycyl-glycine

' Cf. E. Fischer: Ber. 39, 530 (1906).

ALBUMINS OR PROTEINS. 179

In the same way we can conceive a combination of two alanine mole-
cules forming alanyl-alanine; from two leucine molecules we obtain leucyl-
leucine, etc. Emil Fischer has called this whole class of compounds
" peptides." Just as the carbohydrates are divided into mono-, di-, tri-,
or polysaccharides, so Emil Fischer has classified the peptides according to
the number of amino acids participating in the composition of the mole-
cule as mono-, di-, tri-, tetra-, penta-, hexa-, etc. and poly-peptides. He
characterizes them according to the amino acids entering into their com-
position. We can just as successfully unite two or more different amino
acids as we can two similar ones in producing peptides. Emil Fischer
and his students have already produced a very large number of such
chains. As examples of these we mention — Dipeptides: glycyl-alanine,
alanyl-glycine, alanyl-leucine, leucyl-alanine, leucyl-glycine, glycyl-i
tyrosine, glycyl-phenyl-alanin, leucyl-proline, prolyl-leucine, seryl-serine,
lysyl-lysine, arginyl-arginine, histidyl-histidine; Tripeptides: leucyl-glycyl-
glycine, leucyl-alanyl-alanin; Tetrapeptides: dileucyl-glycyl-glycine, tetra-
glycine, dialanyl-cystine, dileucyl-cystine; Fenta-peptides: penta-glycine,
leucyl-tetraglycine, etc. The number of combinations possible by link-
ing these amino acids together is necessarily very great. If we also take
into consideration the fact that all of the amino acids, excepting glycocoll,
contain an asymmetric carbon atom (isoleucine has two of them), the
possible number of isomeric combinations is still further increased. The
number of individual optical isomers, according to van 't Hoff's formula,
is represented by 2 ", in which n indicates the number of asymmetric
carbon atoms which in this case — if we neglect glycine and isoglycine —
is equal to the number of component amino acids.

In order to give a more satisfactory idea of the syntheses of peptides, an
example of each important method will be briefly given.

If glycocoll is converted into its ester, CH2 . NH2 . CO . 0 . C2H5, the
latter goes over into glycine anhydride, a diketopiperazine:
.CH2.CO
NH-^ ^NH

""CO .CH2^
according to the following equation: „„ ^^

2 NHo . CH2 . CO . O . C2H5 = 2 C2HflOH + NIl( ^ ' ^ NH.
Ethyl alcohol CO . CH2

From this substance Emil Fischer ' succeeded in producing the first and
simplest of the peptides, by the action of dilute alkali:
CH^ . CO ^
NH '^ ^ NH + H2O = NHo . CH2 . CO . NH .CH2 COOH.

^ CO . CHa^

Glycine anhydride Glycyl-glycine

Cf. literature E. Fischer: Ber. 39, 530 (1906).

ISO LECTURE IX.

In the same manner, although with more difficulty, alanine hydride
gives us alanyl-alanine; while from leucinimide we get leucyl-leucine.

A second method of coupling the amino acids consists in uniting them
with an acid radical containing halogen, and then replacing the halogen
by an NH2 group. The following may act as an example of this form of
polypeptide synthesis :

In order, for instance, to produce glycyl-glycine, glycocoll is caused to
unite with chloroacetyl chloride. Chloroacetyl-glycine results:

CI . CH2 . CO . CI + NH2 . CH2 . COOH = CI . CH2 . CO . NH . CH2COOH

« , , < ^ '+HCL " '

Chloroacetyl Glycocoll Chloroacetyl-glycine

chloride

If ammonia is allowed to act on chloroacetyl-glycine, we immediately
obtain glycyl-glycine:

CICH2 . CO . NH . CH2COOH + 2 NH3 = NH4CI +
Chloroacetyl-glycine

NH2 . CH2 . CO . NH . CH2COOH.

Glycyl-glycine

We can then take this dipeptide, glycyl-glycine, treat it again with
chloroacetyl chloride, thus adding another glycyl radical to it. Treating
the substance thus formed with ammonia gives us diglycyl-glycine:

NH2 . CHo . CO . NH . CH2 . CO . NH . CH2 . COOH.

Naturally, by following out this same method, we can use other acid
radicals, thus introducing other amino acids. Should we, for instance,
desire to produce alanyl-glycine, we start with glycocoll and bromo-
propionyl chloride, foi'ming «-brom-iso-capronyl chloride, which corre-
sponds to leucine.

It will be noticed that, by following the above method, it is only possible
to extend the chain in one direction, — towards the amine group. It
was, of course, also desirable to add new amino acids to the carboxy side.
This was accomplished by chlorinating the amino acids. If phosphorus
pentachloride be added to an amino acid under definite conditions, the
carboxyl group is changed into the COCl group. The free amino acid also
combines at the same time with a molecule of hydrochloric acid. The
hydi-ochloride of the amino- acid-chloride results:
R . CH . COCl

NH2 . HCl.
The peptides can, naturally, be chlorinated, and the grouping further
extended. By this means we quickly obtain long chains.

ALBUMINS OR PROTEINS. 181

As an example of the formation of a polypeptide by lengthening the
chain at the carboxyl end, we may cite the synthesis of leucyl-glycyl-
glycine from brom-iso-capronyl-glycine-chloride and glycine-ethyl-ester.
The resulting brom-iso-capronyl-glycyl-glycine-ester is saponified ; and the
tripeptide, leucyl-glycyl-glycine, results on treating this with ammonia.
This, itself, can then be chlorinated, and again united with a peptide-
ester, or even with a peptide itself.

We have gone into the subject of the synthesis of the peptides some-
what in detail, owing to the importance of the problem. Synthesis has
always been a great factor in biological-chemical knowledge. By this
means the constitutions of many substances have been determined, and
many debatable questions settled. Synthesis, as we have seen, plays an
even more important part in the chemistry of the albumins. With its
assistance we hope to determine the constitution of the albumin molecule,
and with it, also, we expect to clear up the questions relating to the first
decomposition products, — the peptones.

Most of these syntheses have been carried out with inactive amino acids.
The structure of these peptides is definitely known, depending on the
method of procedure. The subject is not so simple when we consider
its stereo-chemical side. We have already mentioned that all the amino
acids, excepting glycocoll, contain an asymmetric carbon atom. The num-
ber of asymmetric carbons in the polypeptides, therefore, corresponds to
the number of amino acids combined in the molecule — with the exception
of glycocoll. If, for instance, we have a dipeptide of the following general
formula,

NHo . CHR . 0 . CO . NH . CHR . COOH,
* *

it is necessary to have, according to van 't Hoff's formula, on account of
the two asymmetric carbon atoms, indicated by asterisks, four different
active varieties. If we designate the optical antipodes by d and /, the
following forms will be possible: dd, II, dl, Id. Two can produce a
racemic compound (dd-ll) {dl-ld) . If we start with the racemic amino
acids, as has been very generally done, we necessarily expect to obtain
two isomeric inactive compounds. This, in fact, is actually the case
in practice. Other complications also arise, as, for instance, when we
combine a racemic amino acid with an active one; for example, in pre-
paring leucyW-tyrosine. Here we have, on the one hand, d/-leucine,
and on the other Z-tyrosine. In this case we expect two compounds:
a dl- and a H-variety. The relations are, of course, much simpler, if we
employ only active components in the synthesis. In such a case, we
obtain only active peptides ; and if we proceed from those optically
active forms of amino acids, which occur in nature, we must obtain amino
acid chains, which correspond to those occurring in the albumin molecule.
For our requirements the optically active polypeptides are naturally

182 LECTURE IX.

of much more importance than the racemic bodies above mentioned.
We have, therefore, referred to them here, only because it will be necessary
to dwell on them more in detail later, when we consider more fully •
the subject of fermentation. It is clear, that we can never tell, a priori,
whether the polypeptides constructed from racemic amino acids, comprise
the modifications existing in the albumin, or not. It is, therefore, of the
greatest importance for the whole future investigation, that Emil Fischer,
supported by his satisfactory method of chlorinating the amino acids, is
producing polypeptides from active materials exclusively, which he obtains
by splitting the racemic compounds into their optically active components.
We must not forget to mention that the peptide chains, produced from
di-amino acids, and especially the di-carboxylic acids, aspartic and glu-
tamic acids, give much greater opportunities for variation. In the
latter cases the amino acids can attach themselves first to the amino
groups, and then, again, at the two carboxyls, thus producing branching
chains, as can be shown by the following formulse:

COOH COOH

,CH3

CH . NH . CO . CH2 . NH2 CH . NH . CO .(NH2)CH . CHaCH^

CHa

CHz CH.,

I I

COOH COOH

Glycyl-aspartic acid Leucyl-aspartic acid

COOH '' CO . NH . CH2 . COOH

I I

CH . NH2 or CH . NH2

I I

CH2 CH2

I I
CO . NH . CH2 ■ COOH COOH

Aspargyl-mono-glycine

CO . NH . (CH3)CH . COOH

I
CH . NH2

I
CH2

I
CO ■ NH ■ (CH3)CH . COOH

Aspargyl-dialanine
These illustrations may suffice to indicate the many possible combina-
tions which may arise by introducing these dibasic amino acids into the
peptide chains. We will refer here again to a discovery which we have
already touched upon. If we hydrolyze albumin, that is, alter the substance
by addition of water, either by acids, alkalies, or ferments, ammonia will

Cf. Lecture on Ferments.

ALBUMINS OR PROTEINS. 183

be set free. This is especially true in many of the varieties of albumin
from plants. Owing to the vigorous action of the acid or alkali, secondary
products, the humin substances are produced. We might imagine that the
liberated ammonia stood in some relation to the formation of these sub-
stances. This assumption is, however, doubtful, because, on the one hand,
the quantity of ammonia set free bears little relation to the amount of
humin substance formed; and then again, ammonia is produced in especially
large amounts in the hydrolysis by ferments. It would be more nearly
correct to assume that acid-amides are present in the albumins, and, pos-
sibly, in the following form : '

COOH COOH

I CHs I

NH2.CH2.CO.NH.CH >CH.CH2.CH.(NH2) .CO.NH.CH

I CH3 I

CH2 ■ CH2

I I

CONH2 CONH2

Glycyl-asparagine Leucyl-asparagine

These compounds are of especial interest to us on account of the fact
that the reserve albumin, which is stored in plant seeds, is broken down
by fermentation when these seeds begin to germinate, while large quan-
tities of asparagine and glutamine appear in its place. We can, of couree,
also conceive that these acid-amides pre-exist in the albumin. As it is,
these problems are still very much in doubt.

As Emil Fischer has pointed out, the simple amide structure is not the
only possible conception of the grouping of the amino acids in the protein
molecule. There is no reason why piperazine rings should not be present
in albumin. The hydroxyacids, like tyrosine and serine, present another
possibility of combination. They can go over into esters or ether-groups
by intramolecular anhydride formation.

A question of utmost importance to us is: What justification have we
for assuming an anhydride system of linkage for the amino acids in the
albumin molecule? There are various reactions among the members of
the polypeptide group confirming this conception. Many of them give the
biuret reaction. It is naturally of some interest that glycyl-glycine and
triglycine do not give the biuret reaction, while tetraglycine does so in a
very marked degree. It has been known for a long time that the so-
called " biuret-base," which has recently been shown to be the ester of
triglycyl-glycine, gives a very strong biuret reaction. It is very easily
produced when glycine-ester is simply allowed to stand carefully protected
against moisture. Dialanyl-cystine shows a very beautiful biuret reaction.
The higher polypeptides, containing seven or more amino acids, as leucyl-
pentaglycine, give a distinctively red biuret test, whose shade exactly

E. Konigs: Diss. Berlin, 1903.

184 LECTURE IX.

corresponds with that of the peptones from silk. Many polypeptides are
precipitated from dilute solution by phospho-tungstic acid. It is also
interesting to note that difficultlj' soluble amino acids produce polypeptides
which are easily soluble, and that difficultly soluble polypeptides often
instantaneously become soluble on the introduction of another amino acid.
Tetraglycine is difficultly soluble. Leucyl-tetraglycine is easily soluble.
As is well known, the peptones are all easily soluble in water, although
it is necessary to remember that we are here dealing with mixtures whose
components may be capable of keeping each other in solution. The changes
which occur in the taste of these substances is also very interesting: for
instance, when sweet-tasting amino acids are linked together the resulting
product has often a bitter taste. The peptones also, as a rule, taste
distinctly bitter.

We must admit that many analogies exist between the synthetic polypep-
tides and the peptones. We can make no sharp distinction in this direction.
We must not lose sight of the fact that we are comparing a sharply defined
chemical compound with a mixture. The name " peptone " does not
indicate any definite compound; in fact, may not even represent distinctly
analogous cleavage-products of protein. It is much better to assume that
the peptones represent all stages of decomposition between that of albu-
moses and the amino acids.

Although we do not expect at present to obtain positive results by
direct comparison in this way, excellent progress has been made by biological
experiments. It was of the greatest significance that certain polypeptides
were decomposed by the pancreatic ferment' in the identical manner as the
peptones themselves. Thus glycyl-Z-tyi'osine quickly breaks down into its
components, glycocoll and ^-tyrosine. It is also especially interesting to
note that the racemic polypeptides are broken down asymmetrically; that
is, only half of the racemic substance is attacked.^ The following example
is given to illustrate this clearly. If we prepare the polypeptide, alanyl-
leucine, from racemic alanine and leucine, we necessarily expect to obtain,
according to the theoretical conceptions previously considered, four com-
binations, which contain the four active amino acids, I- and rf-alanine and
I- and d-leucine. One racemic compound contains d-alanine and d-leucine
and Z-alanine and Z-leucine (rf-alanine-c?-leucine + Z-alanine-Weucine) .
The second is constructed in the following manner: d-alanine-Z-leucine
+ Z-alanine-rf-leucine. The pancreatic ferment, however, splits only
one of these two racemic combinations. Experience has already taught
us that optically active amino acids in the albumins are split off; hence,
we are justified in concluding, that of the two racemic compounds men-

' E. Fischer and P. Bergell: Ber. 36, 2592 (1903); 37, 2103 (1904). E. Fischer and
E. Abderhalden: Sitzungsber. Akad. Wiss. Berl. 1905; Z. physiol. Chem. 46, 52 (1905).
' For further details, see Lecture on Ferments.

ALBUMINS OR PROTEINS. 185

tioned, the ferment will attack only that specific combination which con-
tains the corresponding optically active amino acid in the albumin. As
d-alanine and /-leucine are formed by the hydrolysis of albumin, the racemic
compound necessarily contains the combination, d-alanyl-Z-leucine. There-
fore the partially hydrolysed racemic body, obtained by fermentation is
rf-alanyW-leucine -I- Z-alanyl-d-leucine. The unchanged portion must be
rf-alanyl-d-leucine + ^alanyl-Z-leucine.

We have intentionally taken this example in order to show the selective
action of trypsin on the large number of polypeptides, on the one hand,
and, on the other, to illustrate the fact that the different behavior of the fer-
ments towards various racemic compounds can be utilized in determining
the configuration of the substance in question.

The results obtained by. fermentation only become conclusive after all
of the different racemic compounds have been subjected to the treatment.
We can only then decide wdiether or not a definite compound is attacked
by trypsin. The relations become much more simple when the active
peptides are subjected to investigation. Even then, however, if a certain
combination of amino acids is not hydrolyzed by trypsin, it does not by any
means follow necessarily that such a combination is not present in albumin.
We have already seen that in the breaking down of the different proteins by
trypsin, different amounts of residues remain which resist digestion strongly.
The albumin molecule evidently contains chains which are not broken
by trypsin.

We already know, and shall later discuss the subject more in detail,'
that the ferments, as a rule, work in a specific manner, and are very strongly
influenced by differences of configuration. The fact that trypsin sphts
the synthetic polypeptides is a strong indication for the assumption that
such anhydride-linked amino acid chains are present in albumin.

Another important proof, that completely agrees with Emil Fischer's
assumptions, is obtained by a study of the behavior of the polypeptides in
the animal organism. The}^ are decomposed in the same manner as pro-
teins, even when injected subcutaneously. Glycyl-glycine is hydrolyzed.
A small part of it appears in the urine as glycocoll.^ Dialanyl-cystine and
dileucyl-cystine are similarly acted upon, and the cystine is consumed in the
same manner as if it were introduced as such into the animal organism.
Glyc}'l-?-tyrosin is likewise completely consumed.^ Finally, it has been
definitely proved for glycyl-glycine, tri-glycine, and alanyl-alanine,* that
the decomposition of these peptides proceeds in the same manner as if the
individual components alone had been introduced.

E. Abderhalden and P. Bergell: Z. physiol. Chem. 39, 9 (1903).

E. Abderhalden and F. Samuely; ibid. 46, 187 (1905).

E. Abderhalden and P. Rona: ibid. 46, 176 (1905).

E. Abderhalden and Y. Teruuchi: ibid. 47, 159 (1906).

186 LECTURE IX.

Glycine anhydride and alanine anhydride are likewise utilized. They may
possibly be decomposed in the intestine, and converted first of all into
polypeptides. Leucyl-leucine is utilized in the same manner as leucine.'

The keystone of the whole proof must be regarded as certainly reached
if we can obtain from albumin itself products analogous to the polypeptides.
This has been accomplished. Its accomplishment was due entirely to
applying the knowledge gained concerning the synthetic polypeptides to the
study of the decomposition of proteins. It was found possible to act upon
proteins in such a way that they did not break down entirely into their
simplest components, — the amino acids, — and, on the other hand, to
carry the decomposition beyond the point of the most complicated cleavage-
products. Such a partial decomposition could be most easily accom-
plished by acting on those albumins on which the usual reagents, acids
and alkalies, and, above all, the proteolytic ferments, have least effect. We
have succeeded in obtaining from silk-fibroin, by a preliminary action of
acid in the cold and a subsequent digestion by pancreatic juice, a polypep-
tide in the form of its anhydride.^ All its characteristics, and especially its
cleavage into d-alanine and glycocoll, as well as its conversion into the
polypeptide, proved that a compound was present which corresponded to
the polypeptide composed of rf-alanine and glycine. We do not err in
designating it as glycyl-d-alanine. The compound was isolated as a
polypeptide-ester and converted into its anhydride by the action of alco-
holic ammonia. By splitting the glycyl- alanine anhydride we naturally
obtain two products depending on the portion of the piperazine ring
attacked; thus, glycyl-d-alanine or d-alanyl-glycine result, as is indicated by
the following formula:

II

CH2— COn^tt
NH ^ /iMn

I^CO— CH-CHs

If the ring is ruptured at I, we obtain, by the addition of water: glycyl-
alanine, NH2 . CH2 . CO . NH . CH , CH3 . COOH. If at II, we get alanyl-
glycine, NH . CH . CH3 . CO . NH . CHg , COOH.

The anhydride itself does not, therefore, give us the answer regarding
the form of polypeptide from which it was produced. We do, however,
know that alanyl-glycine is easily separated into its constituents by trypsin,
whereas glycyl-alanine is not acted upon. We may, therefore, conclude
that the compound isolated was glycyl-d-alanine. Then, again, this same
cleavage-product is formed by acid action — whether by concentrated
hydrochloric, or 70 per cent sulphuric acid. Under these conditions, i.e.

' E. Abderhalden and F. Samuely: Z. physiol. Chem. 47 (1906).
' E. Fischer and E. Abderhalden: Ber. 39, 752 (1906).

ALBUMINS OR PROTEINS. 187

in the absence of proteolytic action, a second polypeptide could be obtained
in the form of its anhydride; namely, glycyW-tyrosine and i-tyrosyl-
glycine. Finally it has been found possible to isolate from elastin,
glycyl4-leucine anhydride' and d-alanyl-leucine anhydride;^ furthermore,
glycyl-d-alanine has been isolated directly from silk,^ and l-leucyl-d-
glutamic acid prepared from gliadin.^ The most important discovery in
this field is undoubtedly the fact that the tetrapeptide - obtained from
silk-fibroin, consisting of two glycine molecules,- one of alanine, and one of
tyrosine, shows properties with which many albumoses correspond. This
proves that the albumoses are not closely related to the proteins; i.e.,
they are not very complicated compounds. In fact, the properties of the
so-called albumoses result from the nature of the amino acids of which
they are composed. In the above case the properties of ^-tyrosine are
evident. It will be well, in the future, to drop the name albumose, and
for the present speak only of peptones which are precipitated by
ammonium sulphate and of those which are not. The more complicated
cleavage-products are not precipitated by ammonium sulphate, while
the I simpler ones, | e.g. tyrosine ' or cystine, are salted out by this
reagent.

There is no doubt that other dipeptides, and especially those with longer
amino acid chains, will shortly be discovered in the same manner. The
train of thought suggested by Emil Fischer's difficult researches concern-
ing the constitution of the albumins has thereby received complete jus-
tification. Where formerly all was darkness, a bright light has suddenly
appeared. It is no longer difficult to picture the whole subject of albumin
decomposition. A whole array of new problems is immediately suggested
by Fischer's investigations. While his successes in developing the chem-
istry of carbohydrates and purines were of tremendous value in advancing
both fields from a biological standpoint, it is doubtless true that his new
efforts, which are of far greater biological importance, will result in great
changes concerning our conceptions of the entire biology of the proteins.
Much darkness, however, still surrounds many questions.

We are still incapable of interpreting the significance of the albumins as
food for the animal organism. We anxiously await the moment when
the fetters will be loosened, which for decades have restricted the progress
of the whole subject of biology. We are deeply interested in all problems
in connection with albumin. Here stands a large group of ferments —
conceptions with no tangible support. The same applies to the tremen-
dous number of toxins, anti-toxins, and allied substances. All investiga-
tors of these various subjects are anxiously awaiting the solution of the

' E. Abderhalden and F. Samuely: Z. physiol. Chera. 47 (1906).
' E. Fischer and E. Abderhalden: Ber. 39, 752 (1906).

188 LECTURE IX.

problem of the constitution of albumin! They all anticipate new impulses
therefrom — new developments; and, above all, new methods. Although
many dreams will not come true and many hopes may be unfulfilled, the
biology of the albumins will, undoubtedly, especially in the narrower sense,
open new fields of effort, and, vi'hen placed upon a satisfactory foundation,
will show great progress.

Let us see now what assumptions concerning decomposition of the
albumins by ferments can be based upon the above observations. We
have seen that the first cleavage-products of albumin are the peptones.
We can easily imagine that the albumin molecule breaks down in the
first place into a series of long chains of amino acids. Even these may
be much differentiated among themselves. It is not necessary that each
of these chains should contain all the amino acids present in the albumin.
These chains then break down into cleavage-products containing a smaller
number of amino acids. We can imagine hereby that a complicated
cleavage-product breaks down into several simpler ones, each containing
more than one amino acid. Many observations indicate, however, that
the amino acids themselves appear at an early stage.

It is of interest that practically simultaneously with the appearance of
tyrosine, cystine, tryptophane, etc., in the digesting liquid, those products
called albumoses diminish in amount, and finally disappear. This cor-
responds to the observations made in the breaking down of tetrapeptides.
As soon as the tyrosine is removed by trypsin, the albumose character
disappears. Tyrosine can be detected within a few hours after the
beginning of digestion. Subsequent decomposition takes place with the
constant production of more amino acid. Smaller chains are produced from
the peptones with larger amounts of amino acids, until finally the greater
part of the amino acid chains are decomposed into their constituents. The
peptones are therefore to be considered as a large mixture of various kinds
of polypeptides. The best distinction that we can make is that only those
polypeptides belong to the peptone class which will give the biuret reaction.
Unquestionably, the term peptone will gradually disappear, and we shall
eventually deal only with chemical individuals.

We shall here refer, as we now do in the synthetic chemistry of albumins,
to di-, tri-, tetra-, and polypeptides. The biuret reaction is only used as a
convenience in indicating the limit of the branching compounds to be
included in the peptone class. The polypeptides which give this reaction
gradually pass over into those which no longer do so. Between the
peptones of the longest chains and the simple amino acids there are con-
tinuous transitory stages.

The decomposition of the proteids by means of trypsin can be illustrated
in the following manner:

ALBUMINS OR PROTEINS,
etc. — Albumin— etc.

189

Polypeptides of high molecular weight which for the most part still
contain all the amino acids

/\ /\ /\

Amino Poly- Amino Poly- Amino Poly-
acid peptides acids peptides acids peptides

. /. I I \

Amino acids Amino acids

/
Amino acids

Decapeptides

Decapeptides
i \

Amino acids

Peptone

Pentapeptide
/\
Tripeptide Dipeptide
/\ /\

Amino Dipeptide Amino Amino acid
acid /\ acid

Amino acid Amino acid

Pentapeptide

I ^ Amino
Tetrapeptide acid
I Amino
Tripeptide. acid

I Amino
Dipeptide^ acid
I Amino
Amino acid acid

This is, of course, only a scheme, and we are frank in stating that
future investigations alone can establish its validity. Many other com-
binations are possible. We can easily imagine that the amino acid chains
also break down in such a manner that the amino acids are not produced
immediately, but that chains are formed, containing only a part of the
original amino acids occurring in the polypeptide. In this connection we
are reminded of dialanylaspartic acid. If we assume that the chain is
lengthened from the alanine group, one of the chains could very easily be
split off, leaving an " aspartic-acid-mono-polypeptide." We must also
remember that there are polypeptides which are evidently not affected by
the digestive ferments. We can very easily imagine such combination as
a result of our investigations concerning the behavior of the synthetical
polypeptides to the pancreatic ferments. It is not without interest, that
the mixture of polypeptides observed in the tryptic digestion of albumin
contained large amounts of phenylalanine and proline, the very acids from
which synthetic peptides were formed that resisted the action of ferments.
If, departing from the plan of our lectures, we attempt here to unravel
a problem which, according to the e.xperimental knowledge at hand, is not
yet fully ripe for discussion, this is done partly because many discoveries
give important support to these views, and largely because only upon such
a foundation are we able to obtain a clearer understanding of the breaking

190 LECTURE IX.

down and building up of albumin in the animal organism. By means of
such a progressive decomposition the cell is able to transform and build
up anew the albuminous material that reaches it, so that it is suited for all
the requirements of the cell-content. It is not difficult to understand how
from a definite compound-albumin all sorts of different proteins may be
prepared containing the various amino acids in proportions quite different
from those in the original mother substance. It is not necessary that
such a transformation should involve a complete reduction of the original
protein to its fundamental constituents; a partial decomposition may
answer all requirements. Although the details of fermentation are
not yet definitely known, we can, however, consider the fundamentals of
protein decomposition as fairly well established. We can also point out
the very close analogy to the disintegration of the polysaccharides. We
know that starch, before it is converted into dextrose, undergoes many
intermediate transformations, concerning the exact nature of which we are,
up to the present time, as much in darkness as we are in regard to the albu-
moses and peptones. We are only able to recognize the former as mixtures,
calling them dextrins. The first definite chemical cleavage-product is the
disaccharide maltose. The dextrins, which we still consider as com-
plicated polysaccharides, correspond to the peptones. The dextrins and
related compounds may very easily be considered as mixtures of long
chains of dextrose molecules. The maltose would then correspond to a
dipeptide. The conditions in the carbohydrates are comparatively simple,
because starch is considered as composed of a series of only one kind of
molecular combination, i.e., dextrose, — whereas, with the albumins, there
are many different fundamental substances. On the other hand, we are
also acquainted with proteins — like the protamine, salmin — which are of
simple construction, being mainly composed of arginine, while we also know
of polysaccharides in the vegetable world which are not far behind the
proteins as regards complexity. As an example of a " mixed " disaccharide,
we have cane-sugar, which breaks down into one molecule of dextrose and
one of Iffivulose, and also to mannorhamnose, which splits into one
molecule of mannose and one of rhamnose.

We are also acquainted with mixed trisaccharides. On hydrolyzing
rhamninose, a glucoside occurring in the fruit of Rhamnus injectoria, two
molecules of rhamnose and one of d-glucose are obtained. Gentianose,
from varieties of Gentiana, contains two molecules of glucose and
one molecule of fructose. We know of a large number of poly-
saccharides, in whose constitution many sugar varieties participate: pen-
toses, methylpentoses, hexoses, etc. We only mention these examples
to illustrate the analogy between the polysaccharides and the proteins.
A very large number of combinations are possible by using many different
constituents. The proteins predominate in the animal organism. They

ALBUMINS OR PROTEINS. 191

are characteristic of the individual tissues. The secret of the individuality
of the various cells undoubtedly depends on their configuration. Every
species, every variety, — in fact, every individual, — has its own " albu-
min." According to this conception, the carbohydrates are of less sig-
nificance to the animal organism. They are essentially food materials,
and are necessarily but a small factor in the production of animal tissues.
It is entirely different when we consider the vegetable kingdom. The
carbohydrates predominate here. They construct the plant tissues, and,
all the numerous living processes are dependent on their presence. Hence
their variety, and their production from the heterogeneous elements.
Carbohydrates, as regards their entire physiological significance and their
composition, are to the vegetable world what the protein substances are to
the animal organism.

The greater the number of amino acids participating in the composition
of a protein, the wider the uses t6 which that protein can be put. On
the other hand, the simpler the function of the protein, the more dominant
becomes one or the other of the amino acids. Fibroin from silk, for
instance, contains 36 per cent glycocoll, and over 20 per cent alanine;
elastin gives us 26 per cent glycocoll, and over 10 per cent leucine; gliadin,
a " reserve albumin " of plants, contains over 30 per cent glutamic acid;
while in the protamines we often find over 80 per cent arginine. It would,
of course, be wrong to compare these albuminous bodies with others, and
say that they were simple in composition. They are simply more homo-
geneous. Whether the amino acids grouped together are all of a kind or
much diversified, has but little bearing on the question of their constitu-
tion or configuration.

The observation that the pancreatic ferment does not attack some of
the synthetic polypeptides, and the discovery that during the digestion of
proteins many complicated cleavage-products remain, which still contain
large percentages of glycocoll, phenylalanine, and ^-proline, leads us to con-
clude that the animal organism utilizes such groups, or similar ones, as a
foundation, or back-bone, for building up new albuminous substances. It
is certainly of some significance that elastin contains so much glycocoll and
leucine. The combination leucyl-glycine, on the other hand, is obviously
unacted upon by trypsin. Silk also contains such compounds, as is shown
by the discovery of glycyl-d-alanine in it. The cell can protect itself by
forming just such combinations. The fact that most vigorous fermenta-
tion processes are continuall}' taking place in the cell, and that in spite of
this the cell retains its own constituents — its amour — intact, becomes
much more comprehensible to us from such considerations.

The significance of Emil Fischer's synthetic polypeptides lies, moreover,
in still another direction. Up to the present time it has not been possible
to test the proteolj'tic ferments as to their homogeneity. We have to

192 LECTURE IX.

content ourselves with a knowledge of their methods of action. There is no
doubt that with the assistance of these synthetic polypeptides, newquestions
will arise in this connection and will probably be solved. We shall be
able to determine whether the various kinds of animals possess the same
kinds of proteolytic ferments, or those which act differently. We are
inclined to the latter behef, at least in special cases. We know that the
feathers of birds and the hair of the Mammalia are sulsjected to the
inroads of parasites, being eaten up by them. These minute animals must
possess much more vigorous proteolytic ferments than have been vouch-
safed to us, because the keratins are, so far as we know, entirely indi-
gestible by vertebrates. We also hope to obtain a complete explanation
of the differences between the action of trypsin and of pepsin by studying
the relations of these proteolytic ferments toward the polypeptides.' Up
to the present time none of the synthetic peptides have been acted upon
by pepsin.- It is possible that the amino acid chains utilized were not
long enough. In fact, pepsin-hydrochloric acid seems to decompose albu-
min in a manner entirely different from that characteristic of trypsin.'
Evidently peptones, and other cleavage-products which do not give the
biuret ' reaction, are produced. There are, however, no amino acids
formed.' The significance of gastric digestion is still quite obscure.
It may possibly be that it causes a preparatory cleavage of the albumins,
so that the trypsin has more opportunity to act; It can also be shown
experimentally that tryptic digestion is hastened and proceeds much
farther, if a pepsin-hydrochloric acid one precedes it.

With the assistance of the polypeptides we also hope to get an insight
into cell metabolism. It has already been demonstrated that the tissues,
especially the liver, contain proteolytic ferments, which are capable of
dissolving bonds between amino acids that are unattacked by trypsin.
Thus, an extract from the liver will split glycyl-glycine completely into
its components.* By extending these investigations to include the various
organs, we will ultimately succeed in finding those which are the most import-
ant factors in the decomposition of the albumins.^ The polypeptides will
also be of great service to us for comparative purposes. It will be of the
greatest interest to learn whether the representatives of the various ani-
mal classes will disintegrate the individual polypeptides in the same manner,
or whether there will be differences in the decomposition products.

From all these problems it is at once obvious how important is the
synthetic linking together of the amino acids into the polypeptides for all
branches of biological science.

' E. Abderhalden and P. Rona: Z. physiol. Chem. 47 (1906).

2 E. Fischer and E. Abderhalden: ibid. 46, 52 (1905).

' E. Fischer and E. Abderhalden: ibid. 40, 215 (1903). F. Obermeyer and E. P.
Pick: Hofmeister's Beitr. 7, .331 (1905).

* E. Abderhalden and O. Rostoski: ibid. 44, 265 (1905).

* E. Abderhalden and Y. Temuchi: Z. physiol. Chem. 47, 1906.

LECTURE X.

ALBUMINS OR PROTEINS.

IV.

Degradation and Foem.\tion of Protein in the Animal and
Vegetable Organisms.

It will be necessary to learn something about the origin of the proteins
in our food, before proceeding to discuss the behavior of such substances
when taken into the animal organism, their decomposition in the alimen-
tary tract, their absorption and assimilation, and the end-products result-
ing from their combustion. The animal organism, as we shall see later,
is only capable of synthesizing its albumins from the same material, or
from its immediate decomposition products. It is incapable of utilizing
inorganic nitrogenous compounds to produce its albumins, and similarly
the animal cells cannot synthesize the albumins from organic nitrogen-
ous substances, unless these are related directly to the albumins them-
selves. The animal organism is entirely dependent on the vegetable
kingdom for its albumin requirements. The plants prepare the proteins
for it.

When living material, whether it be vegetable or animal, decays, its
organic constituents undergo putrefaction. Ammonia, in large amount,
is finally produced from nitrogenous compounds. This is changed into
nitric acid in the soil, nitrates resulting. The formation of saltpeter in
the soil is a process which has been known for a long time. Even H.
Davy ' was aware of the production of nitrates at the expense of ammonia
and atmospheric oxygen. It was eventually discovered that the process
of forming saltpeter, also called " nitrification," was due to the vital
activity of microbes. The pure culture of these organisms followed much
later.^ This was due to the fact that the bacteria possess the peculiar
ability of thriving on an exclusively inorganic nutrient medium, as was
shown by Hueppe ' and Heraeus.*

They satisfy their nitrogen and carbon requirements from ammonium

Elements of Agricultural Chemistry, 1814.
S. Winogradsky; Compt. rend. 110, 1013 (1890).
Tageblatt Naturforscher-Versammlung Wiesbaden, 1887«
Zentr. Bakt. 3, Nr. 13 (1887).

193

194 LECTURE X.

carbonate.' Nitrification is not a simple process. It requires the simul-
taneous activity of several varieties of bacteria. One oxidizes the ammonia
to nitrite, and another converts the nitrite into nitrate. The nitrifying
bacteria are found everywhere. They play an extremely important part
in the economy of nature. They effect the nitrogen cycle.

Even the nitrogen which the animal organism utilizes for its nutrition,
is finally returned to the ground again as ammonia. We shall see later
that the largest part of albuminous nitrogen reappears in the form of urea
in the urine of mammals. Under the action of specific bacteria this is
broken down into ammonia, which is then converted into nitrates. The
plants utilize this anew for the synthesis of albumins, and the nitrogen
completes its cycle of usefulness, first, in the form of inorganic, and then as
organic compounds. This process is not as simple in practice as the
statement indicates. A large amount of free nitrogen is produced simul-
taneously with the combined nitrogen. When nitrogenous organic mate-
rial undergoes combustion, free nitrogen is obtained as well as ammonia.
The amount of the former may be very considerable under favorable
conditions. This is the case when the combustion is carried out at a high
temperature with a liberal supply of air. Nitrogen is also liberated in
large quantity by the explosion of gunpowder. It is liberated not only by
artificial processes, but also through the intermediacy of organisms occur-
ring in nature. To be sure the assumption that free nitrogen is liberated
in the metabolism of higher plants has been disproved by exact investi-
gations, just as the oft-repeated question as to whether nitrogen is elim-
inated as such, from albuminous material in the animal organism, has
been answered in the negative. We are acquainted on the other hand
with a large number of organisms of common occurrence which are incapable
of liberating nitrogen from organic compounds, but can do so from nitrates.
This process, also called denitrifkation, has been known for a long time.

' It may be interesting to note that there are also other organisms capable of utilizing
inorganic instead of organic materials. The group of sulphur bacteria is best known.
Kernels of sulphur are found in their cell-bodies. They thrive in sulphur springs and
produce therein a characteristic flora. They constitute the group of Beggiatoa, and are
aerobic. The Beggiatoa are capable of oxidizing sulphuretted hydrogen, when in the
presence of oxygen, to sulphur. The stored-up sulphur is then further utilized in the
cells, sulphuric acid being formed, which seems to be their characteristic product.
They need only small amounts of organic material. During the oxidation of a gram-
molecule sulphuretted hydrogen to sulphuric acid, 62.4 cal.of heat energy are obtained
by the bacteria.

Many thread-bacteria, especially Leptothrix ochracea, form other examples. They
oxidize ferrous carbonate into a ferric salt, which is decomposed with the formation of
ferric hydroxide. Winogradsky, to whom we owe our knowledge of these sulphur and
iron bacteria, suggests the possibility of the latter participating in the formation of
bog-ore deposits.

ALBUMINS OR PROTEINS. 195

Davy ' called attention to the fact that gaseous nitrogen was set free
from decomposing organic material in the soil. Gayon and Dupetit ^
were, however, the first to announce that the nitrogen originated from
the nitrates. Since that time a large number of bacteria have been isolated
which produced nitrogen from nitrates. Gayon and Dupetit cultivated
two varieties of anaerobic bacteria from the soil, which they called bacterium
denitrificans, a and j3. Denitrifying bacteria can live without oxygen.
They utilize the nitrates as a source of energy. They work, in a sense, in
constant opposition to the nitrifying bacteria. With a liberal supply of
oxygen the latter will predominate, while if the oxygen supply be dimin-
ished, the reverse will be true. There is no doubt but that appreciable
amounts of nitrogen are being continually set free. Nitrogen would be
constantly withdrawn in this way from the organized world were it not
for the fact that other processes are at work to recombine it. We also
know that atmospheric nitrogen and oxygen are combined under the influ-
ence of electrical discharges, producing nitric acid. The amount of nitrogen
combined in this manner must necessarily be small. It is certainly insig-
nificant in comparison with the production of nitrogen from other sources.
During recent years various bacteria have been isolated which possess the
faculty of assimilating, or " fixing," the atmospheric nitrogen. Berthellot '
first called attention to this process. He found an enrichment of soils
which were free from higher plants, and whose only source of nitrogen
was the atmosphere. Winogradsky * was the first to succeed in isolating
a bacterium which was capable of fixing nitrogen directly from the air.
This was the anaerob Clostridium Pasteurianum. Winogradsky showed
that a culture of this bacillus, shut off from every other source of nitrogen
except the air, was capable of assimilating 24.7-28.9 grams nitrogen in
15-20 days. It is interesting to note that this Clostridium is not found
alone, but is accompanied by two aerobic bacteria. Evidently this is a
ease of symbiosis. The aerobic bacteria remove the oxygen which is
deleterious to the development of the Clostridium. They undoubtedly
receive nitrogenous material from the latter in return. Since this dis-
covery other bacteria have been isolated, which possessed the ability of
assimilating free nitrogen. Kriiger and Schneidewind ° describe a bacte-
rium, a culture of which in 62 days converted 4.6-8.5 grams atmospheric
nitrogen into albuminous nitrogen. It is noteworthy that the Clostridium
has been found in the slime of ocean bottoms, and in the plankton of salt

' Elements of Agricultural Chemistry.
' Compt. rend. 95, 644 (1882).

' Ibid. 101, 775 (188.5); 104, 205 and 625 (1887); 106, 569, 1049, 1214 (1888); 107,
372 (1888); 108, 700 (1889); 109, 277 and 417 (1889); 115, 569 (1892); 116, 842 (1893).
* Compt. rend. 116, 1385 (1893); 118. 353 (1894).
' Landwirtsch. Jahrb. 29, 801 (1900).

196 LECTURE X.

and fresh water. The following observations of Kiihn ' are mentioned to
give an idea of the significance of the activity of these nitrifying bacteria.
A field, which for twenty years had not had any nitrogenous fertilizer
added to it, gave an average return of 1976 kilograms of grain per hectare.
Not only was there no decrease in the annual yield due to the gradual
removal of the nitrogen of the soil, but it actually showed an increase of
11.6 per cent in grain produced. There was annually withdrawn from a
hectare of land in crops of rye, from 25-30 kilograms of nitrogen. This
amount of nitrogen must have been taken from the air and transferred to
the soil. Even the fallen leaves in forests assimilate nitrogen by the activity
of the bacteria contained within themselves. It is not at all impossible
that these bacteria are the pioneers in converting decomposed rock into
arable land.

It is a well-known fact that some plants, for instance the legumes,
enrich the soil with nitrogen, while others only deplete it. The practical
farmer utilizes this fact by not planting cereals year after year on the same
soil, but rotates the legumes and the grains. Hellriegel ^ and Willfarth '
have satisfactorily explained the whole subject as a result of their experi-
ments. They proved that the legumes assimilated the nitrogen, and
showed that this formation was intimately connected with the enlarge-
ments of the so-called " root-nodules or tubercles " of these plants. It was
also shown that the legumes could be made to grow nodules on sterilized
soil if infusion of ordinary soil be sprinkled over it. There must evidently
be micro-organisms present in the soil which cause the formation of these
nodules. The infusion obtained from the soil loses its activity on being
heated. The graminw act entirely different. They are not influenced in
their consumption of nitrogen by any infusion from the soil. Their nitrogen
assimilation is dependent on the nitrates already present in the soil. Free
nitrogen is of no service to them. The legumes, on the other hand, are
entirely independent of any increase of nitrates. If the legumes are
grown in sterilized soil, they behave like the graminse. They lose the
faculty of fixing the free nitrogen, and rely entirely upon the nitrates in the
soil. The following experiments are referred to as illustrating the assim-
ilation of nitrogen through the nodules of the legumes. Schloesing and
Laurent * cultivated legumes in sterilized soil and in sterilized glass cyl-
inders. The amounts of carbon dioxide, oxygen and nitrogen in the air

' Friihlings landw. Ztg. p. 2, 1901; quoted by F. Czapek: Bioohemie der Pflanzen:
G. Fischer, p. 131, 1905.

' Tageblatt Naturforscher-Vers. Berlin, 1886, p. 290.

' Tageblatt Naturforscher-Vers. Berlin, Wiesbaden, 1887, p. 362;Zeit. Ver. Ruben-
zuckerind. Beilageheft, 1888, p. 234, and Ber. botan. Ges. 7, 138 (1889). For further
literature see J. Vogel: Zent. Balct. u. Parasitcnkunde 15, 11, 33, 1905.

* Compt. rend. Ill, 750 (1890); 113, 776(1891); 115, 881, 1017 (1892); Ann. Inst.
Pasteur, e, 65 and 824 (1892).

ALBUMINS OR PROTEINS.

197

present were exactly known. Sterilized water was added in one experi-
ment, while the others were watered with infusions of powdered nodules.
Three months later the air was removed from the cylinders and the amounts
of nitrogen present estimated. It was found that it was diminished only
in those cases where nodule extractions had been added. Two of these
experiments in which root nodule infusions were used gave the following
values :

Nitrogen present in air at beginning of ex-
periment

Nitrogen present in air at end of experiment .

Nitrogen absorbed

2681.2 cm.^
2652.1 cm.

( _ 29 . 1 cm.

I 36.5 mg.

2483.3 cm.'

2457.4 cm.
( ^ 25.9 cm.
I 32.5 mg.

The nitrogen absorption can be shown even better by the following
table. In experiment III there were no root nodules present, whereas
I and II contained these:

Nitrogen in the soil and in the leguminous

seeds (peas) at the start

Nitrogen in the soil at the end

Nitrogen taken up by the plants

32.6 mg.
73.2 mg.
40.6 mg.

32.5 mg.

66.6 mg.
34.1 mg.

32.5 mg

33.1 mg

0.6 ma

These root nodules contain bacteria, as has been proved by Beijerinck.»
They live in symbiosis with the cells of the nodules. Beijerinck names the
bacillus B. radicicola. It is widely distributed irt land and water. Recent
investigations indicate that this nitrifying organism is not a separate
individual. It seems as if various bacilli are assigned to the different
varieties of Papillionacce. Successful inoculations of the nodules have only
been possible with closely related members of this family. For instance,
we have not succeeded in forming nodules on the robinia roots by means
of the bacteria from peas. It is also very interesting to note that Soja
Hispida very often fails to produce nodules in European gardens, but will
do so when impregnated with Japanese earth. To indicate the impor-
tance of these discoveries we may add that the nodule bacteria have become
an article of commerce.

It is problematical whether these nodule bacteria are restricted to the
Papillionacce. There are indications that they are also found in other
plant species. They are believed to be present in the Rhinantaccs, Elceag-

' Bot. Zeit. (1888) p. 725.

198 LECTURE X.

nacce, Cycadece, Conijerw, etc. The assumption has also been made that
the hypomycetes, which often exist symbiotically with the roots of higher
plants, have a similar function to that of the nodules. These experiments
have not yet been completed. Many observations on the wild plants
seem to indicate a wider distribution of such symbioses. We know of
many plants which, year by year, always grow in the same spot with the
usual profusion, while many others suddenly spring up and after a short
" period of blossoming " gradually fade away. In this way the dominant
species in a meadow, and especially in a dump-heap, may follow one another
in rapid succession, probably because these short-lived plants rely exclu-
sively on the nitrates of the soil.

It is of great importance to us in considering this subject to realize
that nature possesses ways and means of assimilating the free nitrogen
of the air. On the one hand, nitrogen is set free; and on the other, it is
recombined. We are not prepared to state the relations existing between
these two processes, — whether they maintain an equilibrium, or whether
the liberation of nitrogen far exceeds that of recombination. It would
be very interesting to know the compounds into which these organisms
convert the nitrogen.' At present we have no knowledge concerning this.
We assume that the final substance produced is albumin, which is then, in
part, assimilated by the plants with the help of fermentation.

The discovery that ordinary nitrogen can be directly assimilated closes
the chain of the nitrogen cycle, which had apparently been broken open by
the discovery of the denitrifying organisms. We have forgotten to men-
tion one point. We shall see later, when considering the inorganic nutrient
materials, that the earth possesses the power of " fixing " certain constitu-
ents. This applies, for instance, to the important elements, phosphorus,
potassium, ammonia, etc., which are so necessary for the develop-
ment of the plants. As soon as their solutions come in contact with cer-
tain constituents of the earth they are changed into insoluble compounds,
and are thus protected from being washed away by rain-water. The salts
of nitric acid, the nitrates, behave quite differently. They are not absorbed
by the earth. They are readily soluble in water, and are continually being
washed away, carried to brooks and streams, and finally appear in the ocean.
The amount of nitrogen abstracted yearly from the soil in this manner
is really enormous. K. Brandt- estimates it at 40,000,000 kilograms
per year. We are confronted with the question of how this large amount
of nitrogen is returned to the general nitrogen cycle. That this must take

' Recently it has been found possible to convert technically large amounts of atmos-
pheric nitrogen into its compounds.

' Wissensehaftlichen Untersuchungen. Report of the Kommission zur llntersuchung
der deutschen Meere, 1899 and 1901. Cf. also, E. Schulze: Schweizer. landwirtschaftl.
Zentralblatt. 1902.

ALBUMINS OR PROTEINS. 199

place is evident from the fact that the vegetation which has grown for
thousands of years still continues doing so in the customary manner, in
spite of the leaching of the mainland which has taken place. There is
practically no difference in the behavior of the plants and animals of the
ocean and those of the mainland. Marine plants assimilate carbon dioxide;
this process also requires the assistance of the sun's energy, which accounts
for the absence of plant life at great depths to which the sun's rays cannot
penetrate. Marine plants also require nitrates for the formation of their
albuminous constituents. The fishes in the ocean also obtain their albu-
min ultimately from the vegetable world. Marine vegetation is incapable
of utilizing all of the immense amounts of nitrogen presented to it. Nitro-
genous compounds are set free from dead plants and animals through
putrefactive processes. These are changed into ammonia, which then goes
over into nitrites and nitrates in the same manner as on the mainland.
The ocean occasionally casts large masses of sea-weed on the shores. The
amount of nitrogen from this source is infinitesimal in comparison with that
leached from the soil. It is, therefore, of great interest to learn that the
ocean also possesses denitrifying bacteria, which are continually setting
nitrogen free. They complete the cycle of the nitrogen washed into the
ocean as nitrogen compounds.

The significance of the great value of the denitrification process now
becomes apparent, in contradistinction to its undesired appearance in the
soil. The great part which the nitrogen-assimilating bacteria play in
nature, is now explained. The living requirements of the whole world of
organisms guarantee an interchange of material! These smallest living
beings furnish us with the fundamental requirements of our existence.
The discovery of the denitrifying bacteria has also solved an apparent
contradiction. It is well known that the concentration of plant and
animal life on the mainland diminishes from the equator towards the
poles. This is not the case in the ocean. This circumstance is very strik-
ing, as one would be led to expect much better development of conditions
in the tropical seas, owing to the great preponderance of light, in com-
parison with the dark arctic regions. It is probable that this state of
affairs may be traced to the denitrifying bacteria in some manner. A
tropical environment is ideal for them and their activities. They develop
to best advantage at a temperature of 25°-30°C. They would, there-
fore, abstract more nitrogen from marine plants in tropical seas, than in
the seas of the arctic zone. We will at once state that this is merely offered
as an explanation. We know that the growth of all organisms is governed
by the Law of the Minimum, i.e., of all the substances which are accessible
to the organism, the amount utilized is governed by the amount of that
substance present to the smallest extent. While the marine plants may
have a large quantity of available nitrogen in the form of nitrates, it may

200 LECTURE X.

be that the supply of, say, phosphorus, for example, is unusually small.
The plants would then only be capable of utilizing the nitrates in propor-
tion to the amount of phosphorus present. It is perhaps possible that
the conditions of nourishment are different in different zones.

In any case, the free nitrogen in organic nature plays an exceedingly
important part in the nitrogen cycle. The amounts of nitrogen produced
artificially, whether by the combustion of organic substances or by the
explosion of gunpowder, although large, to Ije sure, have little effect upon
the equilibrium of the nitrogen cycle. Such amounts are in time recom-
bined and again take part in the natural cycle.

Albumin contains, besides nitrogen, also carbon, hydrogen, and sulphur.
We have already pointed out the central position that the carbohydrates
in the plant organism play in the assimilation of carbon dioxide, and called
attention to the fact that it evidently forms the starting point of the syn-
thesis of albumin. In another place we shall go into this matter more in
detail. Here we shall merely suggest that certain relations are known
to exist between the simple carbohydrates and individual amino acids, so
that we can easily understand the formation of the latter from the former.
Thus carbon and hydrogen for the synthesis of albumin originate in the
air and water. In this form the animal organism gives back these elements
to the vegetable kingdom again.

The plants obtain their sulphur from the soil, in which this element is
present as sulphates of the alkalies and alkaline earths. Plants utilize
sulphur almost exclusively for the formation of albumin, and it also reaches
the animal organism in this form. In the animal, this sulphur is largely
converted into sulphuric acid, and given back to the general cycle in the
form of its alkali salts.

It is difficult to estimate the value of allniminous suljstances to the vege-
table kingdom, from the experiments at hand. Our knowledge of the
metabolism of albumin in plants is remarkably slight. We are certain
that it by no means plays the same part in the plant that it does in the
animal organism. We should like above all to know whether the plants
consume albumin at all, i.e., oxidize it. Oxidation processes, as we have
seen in considering the assimilation of carbonic acid, play a subordinate
role in plants to processes of reduction. They undoubtedly take place to
some extent. We do know that the albumin in the animal organism is
almost entirely decomposed, partly into urea, or partly into uric acid. Such
substances have been looked for in vain in the vegetable kingdom.' It is
interesting, however, to learn that many plants produce substances closely
allied to the uric acid group, namely, methylated-xanthine derivatives,
such as theo-bromine and caffeine, both of which are important con-

' A discovery of urea has been reported among the varieties of Lycoperdacew. Cf.
Max Bamberger and Anton Landsiedl; Monatsh. 24, 218 (1903).

ALBUMINS OR PROTEINS. 201

stituents of table accessories. Concerning the relations which the purine
bases, and their accompanying intermediate products, bear to the
metabolism of albumin, nothing can be affirmed. It is, of course,
possible that they are derived from the nucleins. The alkaloids have
often been assigned a relationship to albumin. We know little about
their origin. It is possible that the dyestuff, indigo, is related to albumin
metabolism. In discussing the decomposition products of proteins we
met with tryptophane, skatole-amino-acetic acid, and have seen that
putrefaction decomposes this into indole, skatole, skatole-acetic acid, and
skatole-carboxylic acid. Skatole is rarely found in plants. The Japanese
wood, Ulmacace, Celtis reticulosa Miq., contains approximately 1 per cent
of skatole. Recent investigations also indicate the occasional appearance of
indoxyl among plants. We know nothing definite about the relations of the
indoxyl derivatives to the albumin decompositions in the plant organism.

We are not much better informed concerning the processes participating
in albumin syntheses. The nitrates which the plant takes up must be
reduced. It is now usually assumed that the leaves are mainly instru-
mental in effecting the albumin syntheses; the idea being that amino acids
are first formed, which by recombining among themselves produce higher
complexes, and finally albumin itself. Although the leaves themselves are
incapable of utilizing the nitrogen of the air, as such, observations have
been made indicating that they can absorb small amounts of ammonia.
It seems that light has also an effect upon the production of albumin.
Albumin is, to be sure, formed in the dark, but the synthesis proceeds much
more rapidly in the sunlight. We are still entirely in doubt as to how
the amino acids are formed. We can assume, as previously indicated,
that they are derived from simple carbohydrates, — for instance, from gly-
cerose; or we can just as easily imagine that the formation of the amino
acids is a more direct assimilation process. The manner in which the
nitrates are utilized presents a difficult problem. It is certain that they
must be reduced. The nitrite formation has also been followed directly. We
have assumed that HNO3 goes over into HNO2, and this into HN:0.
The addition of water would produce hydroxylamine, NH2 . OH, which,
in conjunction with formaldehyde, obtained by the reduction of carbonic
acid, produces formamide, HCO . NH2.' The formation of hydrocyanic
acid and the reduction of a nitrate to ammonia have also been suggested.
It is almost impossible at present to draw any positive conclusions. The
synthesis of albumin in the organism of plants, or, perhaps better stated,
the formation of the amino acids, as yet remains entirely unexplained.

We have a better conception of albumin metabolism in germinating
seeds. Ripe seeds contain large stores of albumin. They, therefore, act

' A. Bach: Compt. rend. 122, 1499 (1896).

202 LECTURE X.

as sources for the production of plant proteins. We note great changes
as soon as germination begins; in fact, the entire cell contents are drawn
upon. We have already considered the conversion of the carbohydrates
into fats, and vice versa. Besides such processes hydrolysis undoubtedly
causes other metabolic changes to take place. The proteins are disin-
tegrated into their components by the activity of proteolytic ferments.
Complicated products, " peptones," ' are first formed, and finally amino
acids, which, at least in part, are then decomposed further. We may
compare the beginning of germination with the intestinal processes. The
purpose is in many respects the same. The germinating cell disintegrates,
in order to utilize the various elementary components for the construction
of a new cell body. We are still undecided whether the protein molecule is
completely, or only partially, disintegrated by hydroly.sis. Asparagine
has been detected in germinating legumes, while glutamine has been
observed in other cases. The amount of asparagine may be increased by
germinating in the dark. We are still unaware of the significance of the
formation of asparagine. It is possible that it does not participate further
in the construction of albumin, but that it acts as an intermediate step
to other nitrogenous substances; or, even, that it has nothing further to do
with such substances, but now enters into relations with the carbohydrates
and fats.

That asparagine does not directly participate in the synthesis of albumin,
which immediately follows its breaking down, is evident from the fact
that it does not diminish to the same extent that the albumin formation
progresses.

We will add here that the seedlings at the beginning of their existence
also disintegrate their other constituents, the nucleins, fats, etc., into the
components. It reconstructs everything anew.- We may compare the
metabolism of this germinating seedling with that of the animal.

When we take everything that we know about the formation of the
proteins in the vegetable kingdom and their albumin metabolism into
consideration, we find it very difficult to formulate any distinct conception
of what actually occurs, based on experimental results. We have felt
that we ought to consider briefly this subject here, because, as we have re-
peatedly said, we can expect to have a complete understanding of biological
processes only when we have as broad a foundation as possible. There is
no sharp dividing line between the plant and animal kingdoms. It would
be a gross error to try to separate the biological investigations in these two
fields. The absolute dependence of the animal organism on the products
of the vegetable kingdom forces us to consider in detail the biological
chemistry of plants.

' W. R. Mark: Z. physio!. Chem 42, 2.59 (1904).

2 Cf. among others, J. R. Green and H. Jackson: Pr. Roy. Soc. 77 (B), 69 (1905).

ALBUMINS OR PROTEINS. 203

Let us now return to the relations of the animal organism to the albu-
mins it obtains in its food. They are not at all attacked by saliva, with
which they first come into contact. This secretion does not contain any
ferment which can act upon the proteins.

The albumins are next subjected to the action of pepsin in the stomach.
Spallanzani ' was the first to give a clear demonstration of the digestive
action of the gastric juice. Normal gastric juice reacts acid. It contains
free hydrochloric acid. This was definitely established by Bidder and
Karl Schmidt." They estimated quantitatively the total chloride present
in the stomach, and also all the bases, — potash, soda, lime, magnesia,
iron oxide and ammonia, — and found after computing the amount of
hydrochloric acid required to combine with these, that some remained
uncombined. We shall discuss the composition of gastric juice and its
secretions more in detail later, confining ourselves at present to the state-
ment that the proteolytic ferment mentioned, i.e. pepsin, is only active
when in acid solution. It was first believed that the pepsin was united
with the hydrochloric acid and exercised its functions as pepsin-hydro-
chloric acid. It was, however, soon shown that the hydrochloric acid
could, on the one hand, be substituted by other acids, for instance, lactic
acid, while, on the other hand, other acids did not replace the hydrochloric
acid in equivalent amounts. The amount of hydrochloric acid in the gas-
tric juice is very appreciable. The gastric juice of dogs contains 0.5-0.6
per cent hydrochloric acid; that of cats 0.5 per cent; while for human beings
from 0.2-0.3 per cent is reported. The attempt has been made to assign
to the hydrochloric acid content of the stomach an antiseptic action as
its greatest function. Although there is undoubtedly such an action, the
fact also remains that hydrochloric acid participates in the digestion of
albuminous substances. The mechanism of its activity has, however, not
been thoroughly explained. It may be summed up as follows: If we add
hydrochloric acid, or even gastric juice, to albumin, a peculiar change
takes place. The albumin swells up and fills the entire vessel as a gel-
atinous mass. Large amounts of hydrochloric acid are simultaneously
combined. The quantity of free hydrochloric acid diminishes. We can
imagine that the hydrochloric acid enters into combination with the albu-
min, producing soluble albumins, the so-called " acid-albumins." It is
possible that the hydrochloric acid loosens up the albumin molecule, i.e.,
changes it in some way, preparing it for the action of pepsin. There are,

' Versuche iiber das Verdauungsgeschaft. German by Michaelis. Leipsic, 1785.
Eberle: Physiolog. Verdauung aiif natiirlichera und kiinstlichem Wege Wiirzburg, 1834.
Cf. also Gamgee: Physiologische Chemie der Verdavuing: Leip.sic and Vienna, 1897.
W. Beaumont: Neue Versuche und Beobachtungen iiber den Magensaft und die Physi-
ologie der Verdauung. German by B. Luden. Leipsic, 18.34.

' Bidder and Schmidt: Die Verdauungssiifte u. d. Stoffwechsel, Mitau u. Leipsic,
1852.

204 LECTURE X.

for instance, many albuminoids nearly immune to the action of the gastric
juice, which, under the influence of mineral acids in the cold, are so
changed that they are then appreciably disintegrated by pepsin. It
seems, however, that the hydrochloric acid, besides exercising this effect
on the albumin, also, in some manner, directly influences the pepsin.
This is evident from the fact, that, when all the hydrochloric acid has
combined with the albumin and its cleavage-products in an artificial digest-
ing mixture, the pepsin digestion then ceases, and can only be brought to
renewed activity by the addition of fresh hydrochloric acid. We are
justified in believing that the albumin combines with more hydrochloric
acid, in proportion to the greater number of cleavage-products formed.
Direct observation has also shown that the hydrochloric acid disappears
in proportion to the time of digestion.

The breaking down of the proteins by the action of the gastric juice is
not at all extensive. Complicated peptones are mainly formed, accom-
panied, of course, by some of the lower cleavage-products, evidently of
the group of simple polypeptids. Amino acids, under normal condi-
tions, are not to be detected.* The digestion of albuminous substances
in the stomach evidently serves to prepare them for the action of
trypsin, with which they next come in contact. Test-tube experiments
have shown that tryptic digestion is quicker and more intense when
preceded by a pepsin-hydrochloric acid digestion.^ Certain difficultly
digestible albuminous substances, as for instance serum-globulin, show
this property very plainly. It is evident that the preliminary digestion
in the stomach of the different albuminous substances is of varying
importance. It is of little significance for those easily digested. The
advantage of such a preliminary decomposition becomes especially clear
when we consider how rapidly the absorption of the albumin cleavage-
products follows in the duodenum and the remaining small intestine.
In spite of an extremely liberal diet of meat, we find only small amounts
of digesting material in the duodenum. Accordingly as the stomach
is emptied through the pylorus, trypsin digestion and the absorption of
cleavage-products take place in rapid succession. A much larger field
of action is presented to the trypsin ferment at one time. Instead of
acting upon an albumin molecule, it can immediately attack a large
number of cleavage-products, and quickly disintegrate them into more
simple components.

The proteids, when in the stomach, are disintegrated first of all into
their constituents. Hematin is split off from hemoglobin, and the globin
is digested by itself. The nucleoproteids give off nuclein, which, being

' Emil Abderhalden: Z. physiol. Chem. 44, 17 (1905).

' Emil Fischer and Emil Abderhalden: ibid. 40, 215 (1903).

ALBUMINS OR PROTEINS. 205

only slightly attacked by the pepsin, remains undissolved for the most
part, and for this reason was first discovered.

The pepsin of the gastric juice also possesses another action besides
that of direct disintegration. It causes milk to curdle. This striking
property, not yet entirely explained, is due to a specific ferment, known
as rennin,' or chymosin. We will state at once that the assumption of a
separate ferment has been questioned. Pawlow and Parastschuk - con-
clude, from their experiments, that the two actions attributed usually
to pepsin and rennin are due to the same ferment. They arrive at this
conclusion from the fact that the proteolytic- and milk-coagulating effects
of the ga.stric juice take place parallel to one another. Both actions are
accelerated and retarded by the same influences, not only qualitatively,
but also quantitatively. We know that neither the pepsin nor the rennin
is secreted as such in the walls of the stomach. Both are found in the
inactive form as zymogen. It is only by the action of acid that this zymogen
is converted into active ferment. Activating these ferments of the gastric
juice is another important function of its acid contents. The ferments are
only present in their inactive state, and are incapable of doing their work
when the free acid of the gastric juice is missing. Pepsin and rennin are
both rendered active by the same agent; in fact, to the same extent.
This parallelism has caused the Russian authors above mentioned to
stop speaking of two ferments, but of two different actions of one and
the same ferment. We cannot, at this time, accept Pawlow's decision.
The more we study the action of ferments, the clearer it becomes that they
are delicately adjusted for definite compounds, and are influenced by
very slight differences of configuration. In fact, such differences in the
configuration of molecules are oftentimes first indicated by the fact that
a ferment does or does not act upon a certain substance, whereas its
ordinary chemical behavior has not led us to suspect such a difference.
This of itself is sufficient to make it seem improbable that a ferment
could have two such different actions. An important objection immedi-
ately arises. We do not e.xactly know how to interpret the activity of
rennin. It is, of course, possible that this activity may produce the same
result as does pepsin, namely, an hydrolysis. We know that the essential
function of rennin consists, not, as formerly believed, in the curdling of the
casein, but in the conversion of casein into another albuminous body,
possessing entirely different characteristics. If the assumption be correct
that this is merely a hydrolysis, then the analogy to that of pepsin would
be complete. We would only have to assume that the nature of the

' Cf. O. Hammarsten: Sitzber. kgl. Gesellsch. Wissensch. Upsala. 1877.

' Z. physiol. Chem. 42, 415 (1904), and Die Identitat des Pepsins und Chymosins.
Verhandl. Sektion Anat., Physiol, mediz. Chem. Vers, nordischer Naturforscher u. Aerzte
in Helsingfors. 7, 12, July, 1902, p. 28.

206 LECTURE X.

cleavage-products was due to the peculiar nature of casein, which we will
soon consider. In this case it would be more correct not to speak of a
milk-coagulating function, but of that of the proteolytic ferment, pepsin.
We are inclined to look upon the pepsin and rennin ferment as probably
identical, not from the fact that these have never been isolated in a satis-
factorily pure condition, but more especially because of the interesting
observations of Pawlow and Parastschuk. They found that the secretions
of the pancreatic gland act towards casein in exactly the same manner as
does the gastric juice, with this modification, that the proteolytic ferment
of the pancreatic juice, trypsin, acts only in alkaline, neutral, or weakly
acid solutions. The establishment of this fact has settled one thing. We
must either assume that different ferments exist which will coagulate milk,
i.e., one which acts in distinctly acid reaction, and another which is efficient
in neutral or alkaline solution (the rennin of the stomach and the trypsin
from the pancreas are activated by entirely different agents); or, as is far
simpler, we must assume that only one process takes place, namely, a
hydrolysis. Coagulation occurs as a secondary effect in the general
decomposition of casein. It is caused by the precipitation of the early
cleavage-products. It is possible that this stage of decomposition, which
probably takes place before the formation of peptones, is common to
all proteins. On the other hand, it is also possible that casein occupies
a unique position, and that perhaps, corresponding to its functions, it
represents a particularly complicated protein. We should like to place
stress upon the above observations of Pawlow rather than upon the
established similar behavior of the two ferments, and will state once more
that we do not wish to speak of two different actions of one ferment.
Until we have an accurate knowledge of these relations we will retain
the conception of two separate ferments, pepsin and rennin, in our pre-
sentation of the subject.

Rennin is very widely distributed in the whole animal kingdom, and
ferments acting in an analogous manner are undoubtedly found in the
vegetable world. It has been found in animals which chew their cud,
and especially in the fourth, so-called rennet-stomach of the calf. Its
main function has generally been considered to be the curdling of milk.
It has been found, however, that this precipitation of casein is not the
primary process. The nature of casein is changed first of all by the action
of the rennin. Another protein with different properties is produced.
Curdling depends on the formation of insoluble calcium salts, arising from
the casein, which has been changed by the rennin. That this conception
is correct is evident from the fact that casein will not be curdled by rennin
if the solution is free from calcium salts, but it will, nevertheless, undergo
a change. If the casein, treated in the above manner, is then boiled, —
thus destroving the rennin, — it will curdle on the addition of calcium

ALBUMINS OR PROTEINS. 207

salts. The latter property is therefore dependent on the presence of cal-
cium salts, and has no direct relation with the rennin as such. The albu-
min formed, para-casein, differs essentially from casein, inasmuch as it is
precipitated by calcium salts. The precipitate contains large amounts
of calcium phosphate. We do not know what relation this salt has to
the curdling.

According to the general conception, the pepsin action follows the
precipitation of the para-casein, disintegrating it in the same manner as
it does the remaining proteins. The phosphoric acid portion, the so-
called pseudonudein (a compound as yet insufficiently investigated), is
thrown out at this stage. It does not possess any of the ordinary com-
ponents of the nucleins. The purpose of the rennin action is not at all
clear. It is certainly significant that we always find evidence of the
activity of rennin wherever there are proteolytic ferments. Rennin is found
in the intestine and in the organs. It cannot be denied that the concep-
tion that the activity of rennin is to be regarded as being an hydrolytic
attack, and the precipitation by the calcium salts as only an intermediate
effect, depending on the specific characteristics of the first cleavage-products
of casein, these being then subjected to the further disintegrating action of
the pepsin, is certainly a very attractive one. The behavior of casein
during digestion is thus removed from its special position, while the
assumption of Pawlow and Parastschuk that rennin and pepsin are iden-
tical is given a further support, to be sure in the sense that it is a case of
one and the same kind of fermentation, and not that of two different
actions. These processes are still very obscure.

The opinion has often been expressed that pepsin and rennin are not
simple, individual ferments. The various species of animals are supposed
to possess different kinds of ferments. The rennins from human beings
and from swine are supposed to be different from that obtained from
the calf. Bang ^ has isolated a rennin, parachymosin, from the stomach
of the calf, whose properties are essentially different from those obtained
from other sources. Not even the pepsins from different animals are
alike. Differences undoubtedly exist, which may possibly be due to the
nature of the different nutrient albumins supplied to the animals. As
long as we are not acquainted with the ferments as such, and are unable
to study their activity in uniform materials, it will be difficult to pass judg-
ment on the results obtained with different ferments. It is to be hoped
that the transference of such investigations to the complicated polypep-
tides of known structure and configuration will throw light upon this
subject.

We must refer to another peculiar phenomenon as yet entirely unex-

' Pfluger's .\rch. 79, 425 (1900).

208 LECTURE X.

plained. If some rennin is added to a clear solution of the so-called albu-
moses and peptones, a flocculent precipitate is formed. This is called
plaslein, and is supposed to occur only in the presence of albumoses.
Its amount has been variously estimated, from 3 to 27 per cent. It is
suggested that the closer the cleavage-products stand in relation to albu-
min, the more readily will they be precipitated by rennin. The forma-
tion of plastein is still unexplained. It has been looked upon as a
synthetic process, although this assumption has not been substantiated by
any experimental proof.'

The albumin, already partly disintegrated, passes from the stomach
into the duodenum, being there subjected to the influence of the pancreatic
juice, and especially to the trypsin contained therein. This also is not
delivered to the intestines, as such. It is secreted in the zymogen form,
and only becomes active in the intestine. There is a substance, called
" enterokinase," in the intestinal juice, which converts the trypsin-
zymogen into the active ferment. We shall return to this particular
process later. We were a long time uncertain regarding the extent of
the decomposition of the albumins reaching the duodenum in the form of
a mixture of peptones of different degrees of complexity. We assumed,
until recently, that, as a rule, only the so-called albumoses and peptones
were formed, and that these were absorbed directly. Such a conception is
particularly plausible, if, as has been generally done, digestion is only
regarded as a means of preparing the nutrient material for absorption.
This assumption was still believed, even when free amino acids, especially
leucine and tyrosine, were repeatedly found in the alimentary canal. This
view obtained added support from the experiment of Hofmeister.- He put
a piece of the stomach or intestinal wall of a recently killed animal in a
moist chamber for a time at 40° C, and showed that its peptone content
had diminished when compared with a piece of the same size, whose
peptone content had been immediately estimated. In fact, after two
to three hours, all the peptones had disappeared. Salvioli ' also showed
that peptones quickly disappeared when introduced into a ligated intes-
tine. There seems to be no doubt that peptones are absorbed; in fact,
it has even been asserted that albumin itself is directly taken up. It
was shown by the researches of Kutscher and Seeman," and of O.

' Cf. A. Danilewsky and Okunew: Inaug. Diss. St. Petersburg, 1895. M. Lawrow:
Inaug. Diss. St. Petersburg, 1897. Sawjalow: Diss. Jurjew. 1899, and Pfliiger's Arch.
85, 171 (1901). H. Bayer: Hofmeister's Beitrage, 4, 554 (1903). M. Lawrow and S.
Salaskin: Z. physiol. Chem. 36, 277 (1902). Kurajeff; Hofmeister's Beitrage, 1, 121
(1901); 2, 141 (1902).

' Pfliiger's Arch. 19, 8 (1885).

' Arch. Anat. Physiol. Sup. 1880, p. 95.

* Z. physiol. Chem. 34, 528 (1901 and 1902); 35, 432 (1902).

ALBUMINS OR PROTEINS. 209

Cohnheim/ that the breaking down of the proteins in the intestine was
more extensive than was originally thought. The former succeeded in
isolating crystalline cleavage-products from the intestinal contents of dogs
which had been fed on a diet rich in albumin, the animals being killed
at varying times, — for instance, at intervals of six hours.

The discovery of 0. Cohnheim, that the mucous membrane of the
intestine contains a ferment, erepsin, which further disintegrates the pep-
tones, casts doubt upon the conclusions drawn from the above-men-
tioned experiments of F. Hofmeister and Salvioli.

Recent investigations, from various standpoints, indicate a considerable
disintegration of the albumin molecule. It has been shown for one thing
that intestinal digestion is very similar to that artificially produced by tryp-
sin. Amino acids, e.g., tyrosine, leucine, alanine, glutamic acid, aspartic
acid, lysine, arginine, and histidine, are formed in the intestinal canal and
even the polypeptides which are observed in artificial digestion with
trypsin, and are attacked with difficulty, if at all, by ferments. It is at
present uncertain as to how far the disintegration goes in individual cases,
as to whether polypeptides with a small number of amino acids result, or
that the digestion stops while the chains are more complicated. We
have already shown that we can draw no conclusion as to the extent
of the decomposition simply on account of the appearance of free
amino acids. More complex substances may be present at the same
time.

We have reached a like conclusion concerning the decomposition of the
proteins in the intestinal tract from another standpoint.' The significance
of the function of digestion is not merely to prepare the food for absorption.
It goes far beyond this point. The separate components of the food are
not in a condition suitable for the economy of individual beings. Every
species of animal — in fact, every individual — has its own specifically
constituted tissues and cells. If the diet were alwaj^s the same, the for-
mation of the tissues might bear some close relation to the components
of the food. The diet varies, however, and, especially in the case of
human beings and the omnivora, is exceedingly diverse in nature. In
order to maintain the individuality of the animal, and to make its
organism independent of the outer world in the matter of food taken, it
disintegrates the nutrient it receives, and utilizes those components which
may be of service to it in building up new complexes. This conception
of the process of digestion, as a whole, will become especially clear when
we consider the most important food of growing mammals, i.e., milk.

' IbuJ. 33, 4.51 (1901).

■ Emil Abderhalden: Z. physiol. Chem. 44, 17 (1905); Zent. Stoffwechs.-Verdau-
ungs-Krank. 5, 647 (1904); Med. Klinik. 1, Nr. 1 and 2 (1905); 1, Nr. 46 and 47
(1905).

210

LECTURE X.

All of the tissues are formed from this. The only proteins contained in
milk are lactoalbumin, lactoglobulin, and casein. From these, and also in
the case of animals in which the albumin content of the milk is less prom-
inent than is true of human milk, all sorts of different proteins with
their varying functions must be formed. We need only refer to the albu-
minous substances of the blood, to serum-globulin, serum-albumin, hemo-
globin, then to the numerous albuminous constituents of the tissues, and
all of the other proteins. A glance at the following table will give a good
conception of the great changes which one protein must undergo to produce
all the others.

Glycocoll

Alanine

Aminovaleric acid

Leucine

Proline

Phenylalanine . .
Glutamic acid . .
Aspartic acid . .

Cystine

Serine

Tyrosine

Lysine

Arginine

Histidine

0.9

1.0
10.5

3.1

3.2
11.9

1,2

0.065

0.23

4.5

5.8

4.8

2.6

Serum-
albumin

2.7
present
20.0
1.0
3.1
7,7
3.1
2.3
0 6
2.1

Serum-
globulin.

3.5

2.2

18.7
2.8
3.8
8.5
2,5
0,7

2,5

Globin from
hemoglobin.

4,2

present
29,0
2,3
4,2

r,7

4,4
0,3
0,6
1,5
4 3
5,4
11,0

Glycocoll . . . .

Alanine

Aminovaleric acid

Leucine

Proline

Phenylalanine . .
Glutamic acid , .
Aspartic acid . .

Cystine

Serine

Tyrosine

Lysine

Arginine

Histidine

3,0
3.6
1.0
15 0
2.5
2 0
8 0
2.0

present
3.5

Histon from
thymus
gland.

0.5
3.5

11.8
1.5
2 2
0 5

5.2
6 9
15,5
1,5

25,75
6,6
10

21 4
1,7
3,9
0,8

0,34
0,3

4.7
1.5
0.9
7,1

3,4

3 7
10,0
0,6

ALBUMINS OR PROTEINS. 211

Even if all the proteins so far investigated are not all derived from the
same animal, and the methods of analysis are not so employed as to give
exact results, it is, nevertheless, clear that casein must undergo great
changes in order to make possible the transformation into these very
different products. We have disregarded, to be sure, the other albuminous
components of milk, albumin and globulin. It is possible that certain of
the proteins in the body are more closely related to these than to casein, — ■
at least, as far as their composition is concerned. Such a discovery would
not alter our conception, as there can be absolutely no doubt but that
the casein plays a large part in the economy of the nursling. This is
evident from the large amount present in milk. It might, of course, be
objected that casein is mainly utilized as a combustible material, and
does not participate to any great extent in the building up of the body.
While such an assumption is not yet supported by any proof, still on the
other hand, we can reply that our knowledge of the composition of the
lactoalbumin and lactoglobulin is such as to warrant the belief that they
can only participate to a limited extent in the formation of the albuminous
components of the body. They would also have to undergo great changes
in order to make them available for the requirements of the cells of the
body.

It is not difficult to imagine the formation of all of the varied albuminous
substances from one primitive body, if we take into consideration the
fact that a very extensive decomposition occurs even in the alimentary
tract. From the complicated albumin, the intestine receives the individual
constituents either as such or in long or short chains. The intestine is
able to unite these in varying proportions, forming definite products to
meet its requirements. The same process can take place in every cell.

We might expect to obtain an insight into the digestion of the albumins
by studying the blood. It were conceivable that the cleavage-products
are only recombined in the various organs. Such a conception has much
to commend it. We must not, however, forget that the organism strives
to maintain a constant composition for its blood-stream. The blood has
important functions to fulfill, and any disturbance is accompanied by
far-reaching results. It were, in fact, not a matter of indifference to have
the various decomposition products introduced into the blood. The cells
would require that all the elementary components be brought to them and
in definite proportions, in order that they might build up their own allni-
mins, a condition of affairs hardly probable with a large part of these
components.

We have not yet succeeded in definitely isolating any peptones or
other protein cleavage-products from the blood.* The serum of the blood

' E. Abderhalden and C. Oppenheimer: Z. physiol. Chem. 42, 155 (1904). Cf. also
P. Morawitz and R. Dietschy: Arch. exp. Path. Phann. 54, 88 (1905).

212

LECTURE X.

undoubtedly carries mainly albumin, which occupies the same relation
to the albumin metabolism that grape-sugar does to the transport of the
carbohydrates. As the sugar content of the blood is very constant, so
also, the sum total of the albumins in the blood-serum is subject to but
little variation. The serum-albuminous bodies are mainly composed of
albumin and globulin. Their relative amounts vary. During starvation
the former gradually diminishes, while the latter increases. We could
imagine that the composition of the serum-albuminous bodies were depend-
ent on that of the albumin in the food. This ought to be subject to proof
by direct experiment.' Six liters of blood were taken by venesection
from a horse, which had been fed mainly on hay and oats, and the amounts
of tyrosine and glutamic acid present in the serum were estimated. The
animal was then made to fast a whole week in order to guarantee that the
intestines were entirely emptied of their contents. Another sample of
blood (six liters) was taken, and the amounts of tyrosine and glutamic
acid present in the serum again determined. The animal was now fed an
albuminous substance which possessed 36.5 per cent glutamic acid
and 2.37 per cent tyrosine. The serum-globulin of the horse contains,
under normal conditions, about the same amount of tyrosine, but only
8.5 percent glutamic acid. Serum-albumin contains 7.7 per cent glu-
tamic acid. The animal imder investigation, therefore, was fed an albu-
min, gliadin, which possessed five times as much glutamic acid. The
following table gives a summary of the results:

EXPERIMENT I.

Normal.

.\fter 8 i^aj^s'
fasting.

After feed-
ing 1500 g.
gliadin.

After feed-
ing ISOO g.
gliadin.

Tyrosine

Glutamic acid

Per cent.
2.43
8.85

Per cent.
2.60
8.20

Per cent.
2.24
7.88

Per cent.
2.52
8.25

EXPERIMENT II.

Tyrosine . . .
Glutamic acid

Per cent.
2.50
9.52

After 7 days'
fasting.

Per cent.
2.55
8.52

After feed-
ing 2500 g.
gliadin.

Per cent.
2.48
8.00

E. Abderhalden and F. Samuely: Z. physiol. Chem. 46, 193 (1905).

ALBUMINS OR PROTEINS. 213

These experiments show clearly that the composition of the serum-
albuminous bodies remains unchanged and is independent of the nature
of the albumin administered. The amounts of glutamic acid remained
very constant, in spite of the fact that the horse had to renew a supply of
serum-albumin, due to a large loss of blood. The albumin in the food
must certainly have undergone a complete change before it entered into
the general circulation. The blood was taken from the Vena jugularis in
these e.xperiments. As the albuminous substances do not seem to enter
the general circulation through the lymph-stream, but only through the
blood, it was conceivable that the transformation of the nutrient albumin,
that is, the synthesis of the cleavage-products, was carried out in the
liver. We have, as yet, no exact proof of this. Our present knowledge
indicates that a synthesis of the albuminous cleavage-products takes
place in the walls of the intestine. From the various different albumins
in the food, the serum-albumins are formed first. From the latter, each
cell constructs the protein that it requires. The cell, therefore, obtains
the same nourishment entirely independent of external conditions. The
functions of the intestine and of the digestive ferments are, according to
this conception, to be regarded in a quite particular light. First of all,
they guarantee collectively the correct course of the general metabolism.
The digestive ferments act before the intestine does. They furnish the
intestine with the building materials from which it forms homogeneous
products for the cell-metabolism. It now becomes apparent why any
derangement of the alimentary tract should have such a far-reaching effect
upon all processes of metabolism. It is not the deranged absorption which
is so important. It is the deranged assimilation. The intestine itself is
one of the most important organs. Many important syntheses and changes
are carried on within its walls.

That syntheses play necessarily in albumin-metabolism a part as impor-
tant as in the case of fats and carbohydrates, is evident from the fact that
animals which are supplied with greatly disintegrated albuminous material,
can be easily maintained in nitrogen equilibrium, as the experiment on
the next page shows.'

The casein decomposed by tryptic digestion consisted of 80-85 per cent
of simple cleavage-products, the amino acids constituting, by far, the
larger portion, while the smaller part was composed of substances akin to
polypeptides. At most, 15-20 per cent of the total material administered
consisted of complicated polypeptides, which, however, no longer gave the
biuret reaction. Whether the organism is capable of producing albumins
from the amino acids alone remains undecided, and at present is not sus-

' E. Abderhalden and P. Rona: Z. physiol. C'hem. 42, 528 (1904); 44, lOS (1905).
Cf. O. Loewi: Arc. exp. Path. Pharm. 48, 303 (1902).

214

LECTURE X.

ceptible to direct proof; because, in the fii'st place, we are not acquainted
with all the albumin cleavage-products, and, on the other hand, many of
the amino acids are evidently destroyed during the complete hydrolysis by
acids and alkalies.

Total

Date.

N.

in

Food.

Uri

ne.

Dried

Fa?ces.

N.

Bal-
ance.

Weight

Observations.

KOS.

Amt.

N.

Amt.

N.

Outgo.

Jan. 12

_

_

_

_

_

_

_

fasting.

13

—

—

—

—

—

—

—

—

**

14

2 g.

120

2.30]

0.27

2.57

-0.57

2.740

fed per day:

15

115

2.00|.

14.7

0,27

2.27

-0.27

2.750

33.3 g. sliced meat.

16

130

1.981

0.27

2.25

-0.25

2.785

25.0 g. fat.

17

118

1 46

7.3

0.38

1.84

+ 0.06

2.790

50.0 g. starch.

18

105

1.85,

0.14

1.99

+ 0.01

2.800

10.0 g. cane-sugar.

19

110

1.501

10.0

0.14

1.64

+ 0.36

2.820

5. Og. grape-sugar.

20

2g.

100

1.72,

16.6

0.23

1.95

+ 0.05

2.825

21

u

95

1 . 55j

0.23

1.78

+ 0.22

2.830

22

110

1.38

20.1

0.47

1.85

+ 0.15

2.840

fed per day:

23

115

1.35,

20.4

0.36

1.71

+ 0.29

2.870

23.5 g. digested

24

110

1.39J

0.36

1.75

+ 0.25

2.880

casein.

25

120

1.50,

23.6

0.37

1.87

+ 0.13

2.900

25.0 g. fat.

26

105

1.31J

0.37

1.68

+ 0.32

2.945

50.0 g. starch.

27

100

1.29,

15.3

0.34

1.63

+ 0.37

2.960

10.0 g. cane-sugar.

28

120

1 . 34/

0.34

1.68

+ 0.32

2.970

5. Og. grape-sugar.

29

105

1.39

15 9

0.35

1.74

+ 0.26

2.985

30

100

1.64

12.6

0.19

1.83

+ 0.17

3.010

31

90

1.36|

0.52

1.88

+ 0.12

3.030

Feb. 1

100

1.351

28.8

0.52

1.87

+ 0.13

3.045

2

105

1.51i

20.4

0.38

1.89

+0.11

3.030

3

95

1.53J

0.38

1.91

+ 0.09

3.040

4

110

1.58

16.1

0.45

2.03

-0.03

3.010i

Total

32 g.

_

23.19

_

5.86

29.05

+ 3.01

_

Average

2g.

~

1.45

~

0.36

1.85

+ 0.19

~

Closely related to this question, is the problem whether the organism is
capable of getting along with albuminous substances which are deficient
in specific groups. Under normal conditions we constantly consume a
mixture of proteins. Based on the above conception, regarding the degra-
dation and reconstruction of the albumins, we can imagine that it is imma-
terial whether one or the other protein, or this or that elementary
constituent, is absent. The fact that they are all finally present in the
digesting mixture is the important consideration. We can also imagine
that the animal organism possesses the abilitj' of producing certain amino
acids from others; for instance, glycocoll. That an organism can get
along with an all)uminous substance in which glycocoll is entirely absent,
is evident from the feeding experiment mentioned, in which the cleavage-
products of casein were used.

ALBUMINS OR PROTEINS. 215

The reason that many albuminous substances, Hke the keratins, are not
looked upon as food material, is due to the fact that their amino acid con-
stituents are in such combinations that they are attacked with difficulty,
or not at all, by trj'psin. Among the albuminous substances so far con-
sidered, we mentioned one which was characterized by the absence of
almost all of the members of the aromatic series. This is gelatin. Tyrosine
and tryptophane are entirely absent, while phenylalanine is present in only
very small amount. We are justified in concluding, from the idea pre-
viously suggested, that gelatin is not a satisfactory substitute for albumin
in the ordinary sense. The animal organism seems unable to synthesize
tyrosine and tryptophane. It would have to produce aromatic compounds
from substances of the fatty acid series. It is, in fact, impossible to main-
tain the nitrogen balance by feeding gelatin exclusively. It is, of course,
possible to substitute gelatin for a part of the nutrient albumin, in the
same manner as when we do this with a large supply of carbohydrates or
fats. Taking the above-described metabolic processes into consideration,
we may conclude that gelatin is a much better "albumin-sparer" than
the nitrogen-free foodstuffs mentioned, for the reason that it delivers to
the organism a whole series of albumin constituents which it is capable of
uniting with the other albumin degradation products to form its serum-
albumins. We must expect that it can act as a substitute for more nutrient
albumin, in proportion to the richness of the latter in aromatic groups.
Experiments to confirm this have not yet been undertaken, although
efforts have been made to increase the " nutritive index " of gelatin by
the addition of the missing ingredients, tyrosine and tryptophane. It has
in fact been found possible to increase greatly the amount of gelatin used
as substitute by adding simultaneously these amino acids.' Cystine was
used in these experiments. The reason that we have so far been unable
to replace completely albumin with gelatin, and the addition of the missing
amino acids, may be due to the fact that we are not yet acquainted with
all of the albumin components. It seems far more probable, however,
that gelatin contains numerous combinations, which are very resistant to
the action of the proteolytip ferments, and, possibly, it also restricts the
rapid decomposition and reconstruction in cell-metabolism.

Effort has also been made to obtain values, as albumin-sparers of sub-
stances closely related to albumin, especially of asparagine, which occurs
so abundantly in germinating seeds. ^ The interesting discovery was

' M. Kauffmann: Pfliiger's Arch. 109, 1 (1905). Cf. also the earlier investigations
of Eschle: Vierteljahresschrift naturforsch. Ges. Ziirich, 1876, 36. K. H. Lehmann:
Sitzber. Munchener morphol.-physiol. Ges. March 10, 1885.

2 O. Kellner: Z. Biol. 39, .313 (1900). Politis: ibitl. 28, 492 (1891). S. Gabriel: ibid.
29, 115 (1892). C. Voit: ibid. 29, 125 (1892). Mauthner: ibid. 28, 507 (1891). I.
Munk: Virchow's Arch. 94, 441 (1883). Weiske: Z. Biol. 17, 415 (1881); ibid. 30,
254 (1894). W. Voltz: Pfliiger's Arch. 107, 360 (1905); 107, 415 (1905).

216 LECTURE X.

made that this substance acted differently in the organisms of the car-
nivora than in that of the herbivora. With the former and with the
omnivora, asparagine cannot be utilized as a substitute for albumin ; but
this substance does act as an albumin-sparer with the herbivora. It is
not easy to interpret this result. We cannot exactly realize how asparagine
can act as a substitute for albumin. It is, of course, possible, in fact
very probable, that the animal organism is capable of*forming one amii,io
acid at the expense of another; we cannot, however, believe it possible
to produce albumin from asparagine alone. Such an assumption is entirely
out of the question. It seems more probable that it is active in another
direction. It has been thought that the asparagine in the intestines pro-
tects the albuminous material of the food, before disintegration, against
the attacks of micro-organisms; in fact, it has even been suggested that
the bacteria in the intestines synthesize albumin from asparagine, which
is then absorbed by the organism. It is interesting to note that ammo-
nium acetate ^ and succinamide - are credited with having the same effect
as that of asparagine. We cannot consider this question as solved, from
the investigations at hand; only this much is certain, that asparagine can-
not be looked upon as a substitute for albumin in the sense that gelatin
is. Its action is an indirect one.

In this connection we may state that the lower forms of life, like the
molds, can utilize an individual amino acid as a starting-point in the
synthesis of albumin. This is not so remarkable, for, if the nitrate-
nitrogen is available, then the amino acid-nitrogen ought also to be of
value. Experiments with Aspergillus niger ^ indicate that, within certain
limits, the production of albumin is entirely independent of the source of
the nitrogen. This mold synthesized its albumin just as efficiently in
a potassium nitrate medium as when it was supplied with glycoeoll or
glutaminic acid as its sole source of nitrogen. Furthermore, the exam-
ination of the albumin in the mold showed it to be composed of apparently
the same relative amounts of individual amino acids in all three experi-
ments. Glycoeoll, alanine, leucine, glutamic acid, and aspartic acid were
all obtained from the mold. This phenomenon might lie explained by
assuming that this mold evidently decomposes the amino acids presented
as nutriment, i.e., possibly splits off the amino group, and starting from
ammonia begins the synthesis anew. In the same manner it probably
produces the same substances from the nitrates, and eventually forms
most complicated compounds. Czapek ^ and 0. EmmerUng ^ have already

» O. Kellner: Z. Biol. 39, .3.39 (1900).

' Weiske: Z. Biol. 20, 279 (1884).

» E. Abderhalden and P. Rona: Z. physiol. Chem. 46, 179 (1905).

< F. Czapek: Hofmeister'.s Beit. 1, 542 (1902).

5 O. Emmerling: Bcr. 35, 22S9 (1902).

ALBUMINS OR PROTEINS. 217

shown that the amino acids act very efficiently as sources of nitrogen.
The latter also called attention to the interesting fact that the molds will
only attack the a-amino acids, while other acids, with the amino groups
differently situated, are unacted upon. We may add that Aspergillus
niger will also act on the polypeptides produced from the a-amino acids,
as well as on the polypeptides which are not decomposed by trypsin; for
instance, glycyl-glycin and dileucyl-glycyl-glycin. This is not very unu-
sual, for we know that the animal organism possesses ferments in the cells
which are capable of breaking down compounds unattacked by trypsin.
That this assumption is correct, is evident from the previous description
of the behavior of individual polypeptides in the animal organism.'

Ammonium oxalate - has been observed as a metabolic end product in
mold activity, but only during growth with a supply of certain amino
acids; for instance, glycocoU, alanine, serine, aspartic acid, glutamic acid,
and proline. On the other hand, no oxalic acid is produced from leucine,
phenylalanine, lysine, arginine, and histidine. Ammonia is very often
one of the end-products of mold and bacterial metabolism. We would
refer, for example, to the cleavage of urea by bacterial action with the
formation of ammonium carbonate. The different varieties of mold and
bacteria, moreover, produce unequal amounts of ammonia. Bacillus
mijcoides, for instance, converts as much as forty-six per cent of the
nitrogen in albumin into ammonia.^

We might expect that carnivorous plants, unlike the Aspergillus niger
which we have just considered, would be able to a.ssimilate directly the
amino acids and higher albuminous cleavage-products, synthesizing them
into albumin in the same manner as does the animal organism, i.e. without
prehminary decomposition. The metabolism of such plants has, unfortu-
nately, been studied but little, and we do not even know how they digest
albumin. That there are organisms in the vegetable world which are
only capable of forming albumin from its cleavage-products, is evident
from the researches of Beijerinek,* from which it appears that the conidial-
alga, Cystococcus humicola, the alga of the lichen, Phi/sica parietina, with
which it lives in symbiosis, prepares peptones for the nourishment of the
latter. It would be interesting to study the true parasites and the sapro-
phytes from this point of view.

Let us return to the behavior of the albuminous substances in the
intestine. Not all the cleavage-products of the proteins are absorbed.
A part is decomposed in another manner, and is lost for the further syn-
thesis of albumin in the animal body. Bacteria are present in the intes-

' a. p. 203.

' O. Emmerling: Zentr. Bakt. u. Parasitienkunde, II, 10, 27.3 (1903).

3 E. Marchal: Zentr. Bakt. II, 1, 1753 (1895).

* Beijerinck: Bot. Zeit. 1S90, No. 45, Zentr. Bakt. 13, 308 (1893).

218 LECTURE X.

tines, living at the expense of our food materials and especially the albu-
minous substances. They are found in the small as well as in the large
intestine, and occur there as aerobes and anaerobes.' The intestinal
" flora," that is, the bacterial content of the intestines, is very dependent
upon the nature of the nourishment, and varies with it. The anaerobic
bacteria, and especially the Bacillus putrificus, are largely responsible for
the putrefaction of albumin. The activity of this bacillus is accelerated
by the presence of aerobic bacteria, especially Bacterium coli and laclis
aiirogenes. They predominate when a large amount of oxygen is present ;
the activity of the anaerobes then becomes restricted, as a result of which
the albumin putrefaction is also diminished. When, on the other hand,
there is a small amount of oxygen present, the anaerobic bacteria become
active. That the anaerobic and aerobic species of bacteria act together
is due to the fact that the latter consume the oxygen which restricts the
living processes of the former; while the anaerobes, on the other hand, form
products by their activity which .serve as .sources of nutriment for the
aerobes. Naturally, the action of anaerobic bacteria alone on albumin will
not give the same products as when the two kinds of bacteria act together.
The bacteria themselves are introduced into the intestines with the food.
The intestines of the new-born are sterile.^ No bacteria can be found in
their meconium, the first excretory product from the intestine. When a
definite bacterial flora has settled itself in the intestines, the amount
present is but slightly dependent upon any further addition from the food-
supply. The fate of the bacteria is, then, determined by the food presented
to them, their nutrient medium. There is, furthermore, a vigorous
struggle for existence in the intestines. Those bacteria whose nutrient
requirements are satisfietl most favorably triumph over the others. On the
other hand, a form of symbiosis develops by which one variety of bacteria
will consume the products of another, thus giving rise to quite a variety
of bacteria. Their development is kept within certain limits in a number
of ways, so that the putrefactive processes do not play any great part
in the intestines, and only a small portion of our food is sacrificed to
them.

Great importance has been attached to the hydrochloric acid in the
stomach for restraining bacterial activity; in fact, for a long time it was
believed that this was its most important function. The amount of
hydrochloric acid in the gastric juice varies in different animals, as already
mentioned. It is sufficient, however, to restrict the activity of the putre-

' Cf. also Escherich: Die Darmbakterien des Siiuglings, Stuttgart, 1886. A. Mac-
fayden, M. Nenki, and N. Sieber: Arch. exp. Path. Pharmak. 28, 311 (1891). D. Ger-
hardt: Ueber Darmfaulniss. Ergebnisse der Physiologie (Asher and Spiro), 3, 1, 107
(1904).

' Bienstock; Arch. Hyg. 36, 335 {1890); ibid. 39, 301 (1901).

ALBUMINS OR PROTEINS. 219

factive bacteria, as has been shown by N. Sieber.' Even Spallanzani *
was acquainted with this property. He showed that a lizard which had
been swallowed by a snake did not show any indication of putrefactive
changes during sixteen days in which the digestion of the animal in ques-
tion was carried out. He also found that when he inserted decaying meat
into an animal's stomach that the putrefactive changes were diminished
and that the putrefactive odor disappeared. It is, therefore, certain that
the free hydrochloric acid of the stomach is of service in this direction.
It is, however, questionable whether, as some observers maintain, the putre-
factive changes in the intestines are prevented to any extent by the hydro-
chloric acid from the stomach. At any rate, no increase in putrefaction
has been observed, even after the stomach was completely removed.
Hydrochloric acid is often absent in human beings under certain path-
ological conditions, while, in other cases, it is often secreted in excess. No
definite influence on putrefactive changes is discernible under such
conditions. On the other hand, we should like to call attention to an
indirect significance of the hydrochloric acid of the stomach in this con-
nection. We have already mentioned that the hydrochloric acid un-
doubtedly plays an important part in the preliminary preparation of the
albuminous bodies for their disintegration. This splitting up of the pro-
teins into numerous larger and smaller cleavage-products in the stomach
has for its main object, according to our present conception, the presen-
tation of the largest possible surface of attack to the trypsin, which facili-
tates the further rapid degradation and absorption. The more quickly
these processes are carried out, the less opportunity do the bacteria have
to attack the cleavage-products. That this is the correct assumption,
is evident from the researches of Ortweiler.^ He found that two patients
afflicted with carcinoma of the stomach — a tumorous formation in which
the free hydrochloric acid is generally absent — showed a smaller elimina-
tion of indican after the administration of hydrochloric acid. Indican is one
of the putrefactive products of albumin, as we shall shortly see. That this
retardation is not due to any direct action of hydrochloric acid on the
bacteria is evident from the fact that the administration of pepsin will
have the same effect. The preliminary decomposition of the albuminous
bodies was, therefore, the cause of the diminished putrefaction. The
putrefactive bacteria could not develop their activity in the stomach
under normal conditions. We also introduce air into the stomach with
the food. This is the reason why the aerobic bacteria develop there. They
themselves cannot cause putrefaction. Their action is confined mainly to

' Nadina Sieber: J. pract. Chera. 19, 433 (1879).

' Spallanzani: Experiences sur la digestion. Trad, par Senebier. Nouvelle Mition,
Geneve, 1784; in German: Versuche iiber das Verdauungsgeschaft, Leipzig, 1875.
' Ortweiler: Mitteil. a. d. med. Klinik zu Wiirzburg, 2, 1886.

220 LECTURE X.

the carbohydrates, whose fermentation is an especially vigorous one when
the free hydrochloric acid is absent, as is indicated by the appearance of
butyric and lactic acids in such cases. The antiseptic action of hydro-
chloric acid is, therefore, more to be sought for in this direction.

To the bile, and especially the acid present in it, has also been ascribed
an influence on the putrefactive changes in the intestines. There are,
however, many observations which do not support this view. Friedrich
Miiller ' observed no increase in the amount of indican excreted in a case
of icterus, i.e. an obstruction in the biliary passages, thus preventing the
flowing of bile into the intestine; nor was there any appreciable increase
in putrefactive changes observed in the case of a dog in which a biliary
fistula was made.

The putrefactive processes increase in the intestines only when some
stagnation of the intestinal contents exists. Jaffe, who first called
attention to this phenomenon, also showed that only when a stoppage
occurred in the small intestine did any marked increase of indican appear
in the urine. If we observe any increase in the elimination of indican,
and there is an obstruction in the large intestine, we are, therefore, justi-
fied in concluding that the stoppage reacts back upon the contents of
the smaller intestine. That a stoppage of the large intestine of itself has
but little effect upon the elimination of indican, is evident from the fact
that the greater part of the albumin is absorbed in the small intestine,
while only small amounts, depending on the nature of the food, succeed
in reaching the large intestine. Ellinger and Prutz ^ have determined the
effect of stoppages in various parts of the alimentary tract in a very
ingenious manner. They cut out pieces of the intestine of a dog, and
replaced them so that the oral end of each excised piece was joined to
the distal end of the whole intestine; and, conversely, the distal end was
joined to the portion remaining attached to the stomach. Such a piece
of intestine retains its original peristalsis and prevents the further pro-
gress of the chyme and fseces, in that it continually opposes the activity
of the remainder of the intestine. We shall subsequently take up in detail
the compounds resulting from the putrefactive processes upon the cleavage-
products of albumin.

Z. klin. Med. 12.

Z. physiol. Chem. 38, 399 (1903).

LECTURE XI.

ALBUMINS OR PROTEINS.

V.

Decomposition of Proteins in the Tissues. The End-products
OF Albumin Metabolism.

According to our present conception of the digestion and assimilation
of albuminous substances, we must assume that the different proteins of
our food are converted into the albuminous bodies of the plasma. In this
form the individual cells obtain the nitrogenous material which is abso-
lutely necessary for its maintenance, and, from our knowledge of
metabolism during fasting,' we must conclude that the cells themselves
give up their albumin only in this form to the blood for further transpor-
tation. We know as little about the manner in which the cells produce
their albumin from the proteins of the blood as we do of the reason
why the animal organism, under all conditions, requires such relatively
large amounts of albumin for the proper maintenance of its corporal
existence. In this connection it is especially noteworthy that the growing
nursling consumes as much albumin, proportionately, as does the fully
developed organism. E. Feer' gives 951 grams as the daily quantity of
milk consumed by a boy 29 to 30 weeks old, and weighing 8.23 kilograms.
This amount would contain 15.2 grams albumin, 32.3 grams fat, 58.0
grams sugar, and 1 . 9 grams ash. At this rate a grown-up person weighing
70 kilograms would consume daily:

Albumin 129 grams

Fat 275 ••

Sugar 494 "

Ash 16 "

The following values have been found actually to be the average food
requirement of an adult:

Albumin 118 grams

Fat 56 "

Sugar 500 "

' See Lecture on Metabolism.

» Jahrbuch f. Ivinderheilkunde, N. F. 42, 195, 196.

221

222 LECTURE XI.

The fully developed organism has, of course, losses. We need only
refer to its secretions, to the constant changes of the epidermal struc-
tures, the cells of the mucous membrane, and to all of the observa-
tions regarding the organs themselves, which participate in a continuous
decomposition and reconstruction in the tissues. It is, of course, possible
that these latter processes are much more extensive than we have any
idea, and that new groups are constantly entering the cell contents,
and others leaving. This is in accord with the fact that the animal
organism disintegrates the nutrients to such an extent and adapts them
to all of the bodily requirements. On the other hand, we can form
no clear conception of the manner in which protoplasm and its com-
ponents are used up, nor understand at present why the cells should tear
down its own components in order to make way for the constituents of
the nourishment. As the different cells, according to the tissues of which
they are a part, and the function which they serve, have a specific
structure, and above all possess individualized protein materials, we must
consequently assume that the cells at every moment take their own char-
acteristic liuilding material from the homogeneous mixture of the blood
proteins, and to some extent transform them in a quite complicated
manner. It is possible that the whole phenomenon is an hereditary one.
The single-celled organisms are constantly engaged in multiplying them-
selves. They develop rapidly, producing new individuals in quick suc-
cession. We meet the same phenomenon in some of the more highly
organized invertebrates. From a single individual, thousands and tens of
thousands of new, rapidly growing creatures are formed. The whole cell
material is engaged in constant growth and change. We also find analo-
gous phenomena among the vertebrates. In these cases they are confined
in the grown individual to the sexual organs. Here, also, we observe a
constant increase in the number of cells, and a continual delivery of cell
material, on the one hand eggs, and on the other spermatozoa. The
cooperative efficiency of the other body-cells, in promptly preparing thou-
sands and tens of thousands of new cells, is evident from the tremendous
production of leucocytes at the beginning of any infection. The invading
army is surrounded in the shortest space of time by thousands of white
blood-corpuscles. They form a thick wall, and protect the remainder of
the organism. The leucocytes also play some other, as yet undiscovered,
important role in the animal organism. For instance, they are present in
the intestines in large numbers during digestion. Every one of these
newly formed cells must have a completely-constructed protoplasm. They
must in every case contain all the elementary components; moreover, they
also contain proteins, which alone are capable of giving to their compli-
cated structure its true individuality.

The great facility with which the animal organism produces new cells

ALBUMINS OR PROTEINS. 223

from its tissues may possibly throw some light on the fact that the animal
cell is continuously using up proteins in its economy. Every individual
body-cell is capable of forming new cells, either in renewing its own
structure, or in giving off cells. It obtains the other necessary elementary
constituents from the storehouses of the tissues. Fats and carbohy-
drates are held in reserve. The animal organism has no storeroom for
its albumin. It is only capable of accumulating albumin under very
specific conditions. Even these stores are removed when the nutrition
returns to its normal state. Every cell is evidently restricted to a definite
amount of albumin, to which it closely adheres. If the animal organism
possesses more fat or carbohydrate than it requires, it will store them up.
There is no increase in the metabolism. If, on the other hand, the amount
of albumin administered be increased, then the metabolic changes are
increased. The albumin governs the whole animal organism. We will
add, that from the chemical point of view it is perfectly possible for the
cell, under certain conditions, to produce all its other organic requirements
from albumin. It is possible that it may produce fats and carbohydrates
from albumin. Albumin, from this point of view alone, is therefore a
valuable food-material. Whether such changes actually occur under
normal feeding, that is, with a sufficiency of fats and carbohydrates, is
very problematical, in fact hardly to be assumed.

We will furthermore add that the albumin in the nourishment, which
certainly serves for manifold purposes, may not all participate in the
true cell metabolism. We arrive at this conclusion, from the fact that
the animal organism evidently produces its body albumin from such
differently constituted proteins in our foods. Waste products may very
easily arise. Let us recall the experiment of feeding a horse with gliadine,
which is so rich in glutamic acid. As this animal was only capable of
utiHzing one-fifth of this amino acid for the synthesis of serum-albumins,
the remainder must have been consumed in some other manner. It is
possible that the animal cells possess the ability of producing other amino
acids from a specific one, but we can also imagine that when the nutrient
albumin is transformed into body albumin, many of the elementary com-
ponents are cast aside and directly consumed. We must not forget that
we are now entering into a realm which has so far been but little investi-
gated. We might expect from this assumption that the animal organism
would be satisfied with the smallest amounts of those albuminous sub-
stances whose compositions were closely related to its own proteins. In
this case most of the elementary components would be available. At any
rate, we are not justified in drawing the conclusion that the proteins
in the food take part in the metabolism of the cells from the fact that
the nitrogen — in the case of mammals — reappears in a short time as
urea. We find that amino acids and polypeptides are eventually broken

224 LECTURE XI.

down in the same way as the proteins themselves, although they hardly
ever come into intimate contact with the cells. It has, moreover, never
been found possible to spare albumin by means of a mixture of amino
acids. The animal organism is evidently unable to build up its own
albumin from such material because other essential components are want-
ing.' Why should not groups result under normal conditions which are
unsuitable for further synthesis' and which are, therefore, immediately
deprived of the amino group and consumed ?

The large albumin-requirement of the animal organism would be partly
explained l)y its constantly striving to obtain as much as possible of its
individual building-material. Here also the Law of the Minimum holds,
i.e. the extent of the syntheses taking place depends upon the amount of
that substance which is present relatively to the least extent. Again,
in the formation of the cell material from the serum-albumin, all of the
components of the latter are not utilized to the same e.xtent. Under some
circumstances entire large groups may not be used at all. According to
this conception, it might be that a cell decomposition and replacement,
which of itself was not very extensive, might require a large amount of
proteins. But even with this assumption we are still unable to explain
the fact that a definite amount of albumin is necessary to maintain
an equilibrium in the metabolism. We would expect that the amount of
albumin used would be now more, now less. But the grown organism
maintains its metabolism in a very definite manner, and keeps it, with
very little variation, at a constant level. We ought to get a good idea of
all of these relations by exactly following the course of nitrogen metab-
olism of the salmon during its stay in fresh water. Remarkable trans-
formations occur in its organism. Its muscle-albumins finally produce
the protamines of entirely different composition. Are all of the elemen-
tary components of the former utilized? If this be so, then the animal
organism must be capable of converting one component into another.
These suggestions, needless to state, are insufficient to remove the haze
which still thickly envelops the whole metabolism of albumin.

If we wish to consider this entire problem which is so important to
biology, we cannot neglect any single phase of the whole subject of the
transformation of material; and from this point of view it will be worth
our while to follow the utilization of the nutrient proteins and their value
in the animal organism.

.Although, in considering the questions concerning the assimilation of
the albumin from food, we met with great difficulties, these become appar-
ently unsurmountable when we attempt to follow the proteins till they
are taken up by the cells of the body. The enigmas begin with the

E. Abderhalden and P. Rona: Z. physiol. Chem. 47 (1906)

ALBUMINS OR PROTEINS. 225

cell itself. Here are problems wholly unsolved. We cannot be led away
from this fact by the discovery of more and more nerve fermentation
processes and new cell-ferments. They only suggest the way in which
• the cell disintegrates material, and leave us to imagine the process by
which the cells obtain their energy from the nourishment. We know very
little about their real metabolism.

We must, therefore, give up trying to trace the further behavior of
absorbed albumin in the animal organism, and confine ourselves to the
discussion of the metabolic end-products. We may be able to obtain
some information of cell-metabolism in this way. The albuminous bodies
contain characteristic elements, namely nitrogen and sulphur, which aid
us in recognizing their decomposition products. We know, to be sure, of
other nitrogenous substances besides proteins, which participate in metabo-
lism, and need only refer to the lecithins and nucleins, etc., as examples.
Their amounts, however, are extremely small in comparison with the
proteins, and we are able, from the chemical constitution of the end-
products of metabolism, to indicate definitely their origin. The extent of
the decomposition of proteins in the animal organism varies, depending on
the kind of animal; this at least applies to the final end-products. In
mammals we find the larger part of the nitrogen introduced into the

/NH,
organism reappears in the urine in the form of urea, CO . The amounts

\NH2
are especially large in the case of the carnivora, and less so with the her-
bivora. Only a very small amount of urea occurs in the urine of birds
and reptiles, while it plays a very important part in the economy of the
amphibia and many varieties of fishes. Urea has also been found in the
tissues and the blood of mammalia. It is very striking that the blood
and tissues of many fishes, especially the selachia, contain very large
amounts of urea.' The part they play in the economy of these animals
has not yet been determined.

That urea is one of the end-products in the albumin metabolism, is
evident from the fact that its amount is dependent on the extent of albu-
min decomposition taking place in the tissues. The human urine for
twenty-four hours contains, under normal conditions of nutrition, about
thirty grams urea. The quantity of urea is greater on increasing the
amounts of albumin administered, or by reason of an increased decom-
position of the albumin; and, conversely, any diminution of changes in
the albumin will produce less. This applies only within certain limits.
In many cases an increased disintegration of albumin is accompanied by

» W. V. Schroder: Z. physiol. Chem. 14, 576 (1890). Cf. also O. Hammarsten:
Z. physiol. Chem. 24, 322 (1898). S. Baglioni: Zentr. Physiol. 19. 385 (1905). V.
Diamare: ibid. 19, 545 (1805).

226 LECTURE XL

a small, or even no, increase in the amount of urea in the urine, for the
reason that other nitrogenous decomposition products are also formed,
which, in part, are direct indications of incomplete combustion of the
albumin. We observe an increased albumin disintegration during many
pathological processes, such as fevers, phosphorus, arsenic and antimony
poisoning, and also with an insufficient supply of oxygen. We shall see
later that an increased elimination of nitrogen follows the administration
of the thyroid gland, and of the hypophysis, or of extracts obtained from
these organs. In Morbus Bascdowii we shall become acquainted with a
disease, which is evidently related to the changes in metabolism of the
thyroid gland, and is characterized by an increased disintegration of
albumin.

We are here chiefly interested in this question: How is urea produced
from albumin? We cannot give a direct answer. We may in fact add
that the formation of urea from albumin has never been satisfactorily
explained. We are accustomed to assume that a hydrolytic cleavage pre-
cedes the oxidation of the absorbed and assimilated nourishment. The
cell evidently does not consume glycogen, but d-glucose, and perhaps not
even this directly, but only after the cell has altered it in a manner as yet
undetermined so that oxygen can attack it. We assume likewise that the
fats are decomposed into their components and are then completely oxi-
dized. Many observations indicate that the proteins in the cell-metabo-
lism are first hydrolyzed and then the cleavage-products are consumed.

An observation by Drechsel ' seemed to place the proteins in an excep-
tional position. Drechsel obtained urea from albumin simply by hy-
drolysis, although the yield was very small. We know to-day that the
amount of urea thus formed is dependent on the quantity of arginine
present in the protein.

A. Kossel and H. D. Daldn ^ have also recently shown that urea may be
obtained from arginine in the tissues. They permitted erepsin to act on
clupein sulphate, and found that this protamine was attacked by the
ferment. After a time all of the arginine present in the molecule was
found in the digesting fluid. A repetition of the experiment, using another
erepsin preparation, failed to remove the biuret reaction, even when the
process was allowed to continue through a long period of time. Higher
complexes remained unattacked.

On analyzing the digesting mixture Kossel and Dakin found the follow-
ing products; (1) protone, the peptone of the protamine; (2) arginine; (3)
ornithine; (4) urea; and (5) amino-valeric acid. This discovery indi-
cates that the erepsin further hydrolyzes a portion of the arginine into

' Ber. 23. 3096 (1890).

' Z. physiol. Chem. 41, 321 (1904); 42, 181 (1904). Miinchener med. Wochenschrift
No. 13, 1904.

ALBUMINS OR PROTEINS. 227

its components. These investigators also succeeded in showing that the
sphtting off of urea was due to the action of a specific ferment which they
called arginase. This ferment has been isolated from the liver, kidneys,
thymus and lymphatic glands, and the chopped-up intestine of the dog.
Small amounts are also present in the blood and muscles, but it is alisent
in the suprarenal glands, in the spleen, and in the pancreatic juice. The
formation of urea from arginine proceeds according to the following scheme:

/NH2

C = NH NH2

\ I

NH . CH2 . CH2 . CH2 . CH . COOH + H2O

Arginine
NH2 NHo NH2

= C = 0 + CH2 . CH2 . CH2 . CH . COOH
^NHa " • '

Urea Ornithine (Diaminovaleric acid)

Now arginine is formed in the alimentary tract by the action of trypsin,
and we can easily imagine that this diamino acid is also formed in the
tissues by the breaking up of the proteins. One of the sources of urea is,
therefore, explained by this discovery. But the proteins used as food-
material contain far less arginine than they do of protamines. Only the
smallest part of urea present in the urine can originate from this source,
as the following table shows:

100 gms. Albumin
contains gms. Arginine.

Salmine

Histon from the thymus glands

Gliadin

Gluten-casein

Casein

Edestin

Gelatin

87.4
15.5
3.4
4.4
4.8
11.7
7.6

Another interesting discovery accompanied the conclusion that urea
was split off from arginine in the tissues ; namely, the formation of ornithine.
Jaffe had already proved that as a matter of fact this diaminovaleric acid
is formed in the organism, by showing that ornithuric acid was excreted
when benzoic acid was fed to birds. Ornithuric acid is the dibenzoyl
derivative of ornithine, and has the formula C5Hio(C6H5CO)2 . N2O2.
The intermediate product of metabolism, ornithine, is undoubtedly

228 LECTURE XI.

protected against further oxidation by its combination with benzoic acid,
and is excreted as such.

The question now arises regarding the manner in which urea is formed
from ornithine, and the other amino acids. We can say that it has been
shown that urea is actually formed from amino acids in the animal
organism.' This discovery is of great significance. It had always been
thought possible that the proteins were broken down, not through the
amino acids, but in some other, still unknown, manner, and that the secret
of urea formation was hidden somewhere in such decomposition. If we
administer glycocoll, alanine, leucine, glutamic acid, aspartic acid, aspara-
gine, or arginine, to a mammal, either by the mouth or subcutaneously, we
find an increase of urea in the urine corresponding to the amounts of nitro-
gen added in the form of amino acids. We observe the same result when
we use the polypeptides instead of the amino acids, e.g. glycyl-glycine,
diglycyl-glycine, alanyl-alanine, and leucyl-leucine.^ Even the anhydrides
so far investigated, glycine anhydride and alanine anhydride, are disinte-
grated by the organism of the dog into urea. An especially interesting
case is that of arginine, already referred to, which is composed of two parts,
guanidine and diaminovaleric acid (ornithine). We have already called
attention to the ease with which urea is formed from the first sub-
stance. W. H. Thompson has shown ^ that when arginine is administered
to a dog, the nitrogen of the first component, guanidine, quickly reappears
in the urine as urea. Ornithine itself is more slowly converted into urea.
Even 100 per cent of the nitrogen of arginine can be found in the urine of
dogs as urea. Very appreciable differences sometimes occur in different
individuals. S. Salaskin * has shown that the liver probably plays an
important part in the production of urea from the amino acids. He passed
glycocoll, leucine and aspartic acid through a dog's liver and found that
the blood used as a circulating medium showed an increase in its content
of urea. His method of proof is not very satisfactory, owing to the fact
that his method of estimating urea is not free from criticism.

Before discussing the statements which give us an idea of the chemical
processes participating in the formation of urea, we wish to mention those
hypotheses, which, being based upon experimental results, will give us the
best conception of the formation of urea from albumin and its cleavage-
products. We find it necessary to add that it is extremely difficult, from

' 0. Schultzen and M. Nencki: Ber. 2, 566 (1869), and Z. Biol. 8, 124 (1872). M.
Nencki: Ber. 6, 890 (1872). W. Knieriem: Z.Biol. 10,264 (1874). E. Salkowski: ibid.
4, 54 and 100 (1880).

' E. Abderhaldcn and Y. Teruuchi: loc. cit. E. Abderhalden and Franz Samuely:
loc. cit. E. Abderhalden and B. Babkin: Z. physiol. Cham. 47 (1906).

' J. Physiol. 32, 137 (1905), and 33, 106 (1905).

< Z. physiol. Chera. 25, 128 (1898).

ALBUMINS OR PROTEINS. 229

the literature at hand, to draw a correct conclusion regarding the subject.
In very many cases we have contented ourselves with a more or less
exact method of determining the urea or its antecedents. This proof is
generally an indirect one. We shall, therefore, confine ourselves to the
most important investigations, abstracting only the essentials from each
of them.

M. Nencki ' characterizes the urea formation as analogous to a type of
synthesis, which takes place to a considerable extent in the animal organ-
ism; namely, the combination of two substances with elimination of water.
He uses the formation of hippuric acid from glycocoU and benzoic acid, as
an example:

CfiHs . CO .iOH + HiNH . CH2 . COOH = CeHsCO . NH .CH2 . COOH + H2O.

Benzoic acid GlycocoU Hippuric acid

Again,

C23H39O3 . COOH + H . NH . CH2 . COOH

Cholic acid GlycocoU

= C23H39O3 . CO . NH . CH2 .COOH + H2O.

Glycocholic acid.

We can add other innumerable examples to these. We need only refer
to the formation of glycogen, fats, and albumin from their components,
and to the numerous examples of the conjugation of glycocoU, glucuronic
acid, and sulphuric acid, with other foreign bodies. Thus, we have as aa
example of each :

CgHsOH + HO . SO3K = CfiHs . 0 . SO3K + H2O.
Phenol Pot. phenolsulphate

C10H7COOH + NH2 . CH2 . COOH=CioH7CO . NH . CH2 . COOH + H2O

Naphthoic acid GlycocoU Naphthuric acid

(CH3)3.COH + CgH.oOy = (CH3)3 . CO . CgHgOe + H2O.

Trimethyl- Glucuronic Conjugated Glucuronic acid
carbinol acid

M. Nencki considers the formation of urea from ammonium carbamate
to take place similarly by the ehmination of water:

NH2 . CO . 0 . NH4 = NHo . CO . NH2 + H2O.

Ammonium- Urea

bamate

Ber. 5, S90 (1872).

230 LECTURE XI.

Schmiedeberg ' is of the same mind, only he believes that the urea is
produced from ammonium carbonate. We can imagine that the ammo-
nium carbamate is an intermediate product, as indicated by the following
formulae:

/0(NH4)

/0(NH,)

/NH2

C= 0 ^

C= 0 ->

C = 0 + H2O

^0 (NH4)
Ammonium
carbonate

^NH2

Ammonium
carbamate

^NH2

Urea

Consequently no important difference seems to exist between the last
two views. Hoppe-Seyler ^ and Salkowski ' look upon the formation of
urea in another manner. They assume that cyanic acid and ammonia are
first produced from albumin:

NH2

NCOH + NH3 -> CN . O . NH4

Cyanic acid Ammonium cyanate

F. Hofmeister * has finally proposed a third hypothesis. He assumes
that urea is produced l)y an oxidation synthesis. From this point of
view, a CONH2 group is formed by the oxidation of albumin or its amino
acid, which, at the moment of its formation, unites with the NH2 residue
of ammonia, when the latter is oxidized, thus producing urea.

Of the three theories mentioned, that of Hoppe-Seyler and Salkowski
seems to us the least probable. It has the least experimental support.
Again, cyanic acid has not been discovered in the organism. The Nencki-
Schultze-Schmiedeberg and the Hofmeister hypotheses, on the other hand,
have many observations substantiating their claims. In the first place,
numerous experiments have established the fact that ammonium carbonate,
and all those other ammonium salts which are capable of being converted
into it in the tissues, are changed into urea by the animal organism.^
This applies to the carnivora as well as to the herbivora. It had seemed,
it is true, as if the former were an exception. After the administration of
sal-ammoniac, NH4CI, to rabbits, the increased elimination of urea corre-

' Arch. exp. Path. Pharm. 8, 1 (1879). y

' Physiologische Chemie, Berlin, 1881, pp. 809 and 810.

' Z. physiol. Chem. 1, 1 and 374 (1877).

• Arch. exp. Path. Pharm. 33, 198 (1894); 37, 426 (1896).

» W. V. Knieriem: Z. Biol. 10, 26,3 (1874). E. Salkowski: Z. physiol. Chem. 1, 1
(1877). Cf. also L. Feder: Z. Biol. 13, 256 (1877). I. Munk: Z. physiol. Chem. 2, 29
(1878-79). E. Hallervorden: Arch. exp. Path. Pharm. 10, 124 (1879). F. Walter: ibid.
7, 148 (1877). Corander: ibid. 12, 76 (1880). J. Pohl and E. Munzer: ibid. 43, 28
(1900).

ALBUMINS OR PROTEINS. 231

sponded exactly with the amount of nitrogen added in the form of sal-
ammoniac. The results were not so definite with human beings and dogs.
A part of the ammonia appeared in the urine, and, from the uncertain
increase of urea, it remained undecided whether this was due to the am-
monia diet or an increased disintegration of albumin. The cause of the
different behavior between the carnivora and the herbivora was soon dis-
covered. It depends on the following: The food of the herbivora yields
an alkaline ash, and during its combustion in the organism it forms potas-
sium carbonate which can react with ammonium chloride. Ammonia
is liberated, and can then be utilized for the production of urea. The
conditions are entirely different with the carnivora. Its food furnishes
an acid ash. The hydrochloric acid is not separated from the ammonia
ib the tissues, and consequently the latter is not available for the produc-
tion of urea. If, on the other hand, we feed some ammonium carbonate
to a dog, we likewise observe an increase of urea. These experiments,
therefore, indicate that the organism of mammals is capable of utilizing
ammonia for the production of urea.

The observations of N. Nencki, J. Pawlow, and J. Zaleski ' have indicated
the probability that ammonia normally — i.e. without being artificially
administered — participates in the formation of urea. They showed that
portal blood contained much more ammonia than did the venous blood of
the Uver. The intestine evidently sends ammonia to the liver, where it is
transformed. If the liver be extirpated, we no longer observe any dif-
ference between portal blood and that obtained from any other part of
the body.

W. V. Schroder ^ has shown that the liver can produce urea from am-
monium carbonate and ammonium formate. He passed blood, to which
he had added these ammonium compounds, through the liver of a clog, and
could soon detect an increase in the amount of urea therein. There can,
therefore, no longer be any doubt that the liver plays a very important
part in the production of urea. Analogous experiments were carried out
with the kidneys and muscles, without, however, showing any increase in
the formation of urea in these organs.

That the liver is not to be looked upon as the only place where urea is
formed, is evident from the fact that its production is continued, even if
less in amount, after the liver has been entirely extirpated. These dis-
coveries only became possible after it was known how to make the so-
called " Eck's fistula." ' Mammals do not tolerate the complete extir-
pation of the liver. They die shortly after the operation. They can,

' Arch. exp. Path. Pharm. 37, 26 (1895).
' Arch. exp. Path. Pharm. 15, 364 (1882); 19, 37.3 (1S85).

" M. Hahn, O. Massen, M. Nencki, and J. Pawlow. Arch. exp. Path. Pharm. 32,
161 (1892).

232 LECTURE XI.

however, be kept alive for a considerable time, if the liver is cut off from
the general circulation while the portal vein is sewed to the Vena cava
inferior near the hilus of the liver, and communication then established
between the two veins. The blood then passes directly from the intestine
into the general circulation. The experimental results obtained after
this operation are not always uniform, owing to the fact that some of
the blood will often find its way through the liver on account of a collateral
circulation which may develop.

If, thus, the utilization of ammonia in the formation of urea has been
established, we must now determine whether it is to be assumed that all,
or at least the greater part, of the amino groups present in the tissues are
split off as ammonia, and thus take part in the production of urea. Such
an assumption has much in its favor. In recent years a number of pro"-
cesses have been discovered in the animal organism which indicate the
presence of ferments which cause the removal of the amino group, and,
in fact, such processes are known in the vegetable as well as in the animal
kingdom. We can imagine that entirely analogous to the breaking down
of carbohydrates and fats in the tissues, the proteins are first hydrolysed
with the formation of the separate amino acids, from which ammonia is
split off. Nitrogen-free carbon chains would then remain, by the com-
bustion of which the cells could then obtain their energy. Unfortunately,
we know nothing further about these carbon chains. It is possible that
they enter into relations with the carbohydrates and with the fats. The
CO group for the production of urea does not necessarily have to orig-
inate from the albumin itself.

We have already called attention to the assumption that carbamic acid
may occur as an intermediate product between the amino acids and urea.

It is not known as the free acid: CO , but as its ammonium salt.

^OH

^ NH2 ^ NH2

The close relations between carbamic acid, CO , and urea, CO , are

^OH ^NHo

evident without further comment. We may consider urea as the amide
of carbamic acid. It is difficult to decide, from the investigations at hand,
whether this acid is a normal metabolic product. There are many obser-
vations at hand which indicate that carbamic acid is present in urine, — in
fact, normally so. Abel ' states that he has succeeded in obtaining this

' E. Drechsel and J. J. Abel: Arch. Anat. Physiol. 1891, 236. J. J. Abel and A.
Muirhead; Arch. exp. Path. Pharm. 31, 15 (1893). Cf. also E. Drechsel: J. pract.
Chem. 12, 417 (1875); 22, 476 (1880).

ALBUMINS OR PROTEINS. 233

acid in large amounts from the urine of human beings and dogs after the
administration of milk of lime. Furthermore, it has been observed that
dogs with an Eck's fistula showed severe indications of poisoning, and that
the same symptoms could be obtained by the introduction of carbamates
into the blood-stream. This method of proof is not convincing. Macleod
and Haskins ' have recently indicated the normal presence of carbamates in
the urine and of an increased appearance after extirpation of the liver.
We must admit that the estimation of the carbamic acid was only an
indirect one. On the other hand, the formation of carbamates corre-
sponds very well with the assumption of the production of urea from
ammonium carbonate, as previously mentioned. We wish to emphasize,
however, that its normal occurrence and its relations to the production of
urea have not yet been absolutely proved.

It will be appropriate at this point to call attention to the observa-
tion of M. Siegfried,- which may have some bearing on the formation of
urea as indicated above. M. Siegfried found that when carbon dio.xide
was led into a mixture of equal parts of normal glycocoU and baryta
solutions no immediate precipitation of barium carbonate appeared, as
was to be expected, but the mixture remained clear for some time. It
gradually became turbid on standing. In studying this subject further,
Siegfried found that salts of the following type were formed :

R— n:;

I ^COOH
COOH

Siegfried analyzed several of these salts, for instance, calcium glycocoll-
carbonate (calcium carbamoacetate) :

CH.,— N— C^OO

COO— Ca/

Also the calcium alanine-carbonate (calcium carbamopropionate) :
CH3

I/"

C— NH— COO

COO— Ca/

The composition of calcium leucine-carbonate is:

(CH3)o . CH . CH2 . CH . NH . COO

\ I

COO— Ca

Am. J. Physiol. 12, 444 (1905).

Z. physiol. Chem. 44, 85 (1905); 46, 401 (1905).

234 LECTURE XI.

We will only refer here to these interesting investigations, and await
further developments.

We must also consider Hofmeister's hypothesis and its claims. Hof-
meister succeeded first of all in producing urea by the oxidation of albu-
min, and then also of amino acids, in the presence of ammonia. From 10
grams glycocoll he obtained .3 grams urea. The assumption of an oxidizing
synthesis in the formation of urea has much in its favor. On the one
hand, the conditions in the animal organism are very favorable for such a
production of urea from amino acids; and then again, the whole process
harmonizes very well with our conception of the degradation of the proteins.
The hypothesis, however, has not yet been proved.

If we take everything that we know about the formation of urea in the
animal organism, into consideration, we can conclude that a part of urea
is directly produced by the hydrolytic cleavage of albumin, the amount
depending on the nature of the latter. Arginine is the only source so far
known for this process. This does not, however, exclude the possibility
that other analogous complexes to that of this amino acid may be present
among the as yet unknown elementary constituents of albumin. We
also know that amino acids and polypeptides, when incorporated in the
animal organism, go over into urea. We do not, however, know the manner
in which this further decomposition is accomplished. It is not impossible
that the anhydi'ide formation, or the oxidizing synthesis, plays an impor-
tant part. Both assumptions are supported by experimental evidence. As
far as the place of formation of the urea is concerned, we are certain that
the liver produces it. We do not know whether other organs also par-
ticipate in its formation.

It has been desired to draw definite conclusions from the presence of the
following compounds in the urine. If we administer aminobenzoic acid
to the animal organism we will find carbaminobenzoic acid in the urine:

NH2 . C6H4 . COOH > NH2 . CO . NH . C6H4 . COOH.'

After the administration of ethylamine (carbonate) we find ethylurea:

C2H5NH2 ' C2H5 . NH . CO . NH2. '

Taurine, under analogous conditions, goes over into carbaminoisethionic
acid, sulphanilic acid into sulphanilcarbamic acid, and 0- and p-amino-sal-
icylic acid into the corresponding carbamino acids.

We can easily imagine a conjugation of urea with the compounds in
question, and ascribe an analogous role to it, as to glycocoll, sulphuric

" E. Salkowski: Z. physiol. Chem. 7, 93 (1883-83). Cf. also R. Cohn: ibid. 17, 274,
292 (1893).

' O. Schmiedeberg: Arch. exp. Path. Pharm. 8, 1 (1877).

ALBUMINS OR PROTEINS. 235

acid, and glucuronic acid. The three last compounds unite with a large
number of other substances, as is well known, thus protecting the tissue-
cells from being attacked. Effort has been made to utiUze the above
observations as proof of the occurrence of cyanic acid, while, on the other
hand, Hofraeister has looked upon it in the light of his assumption of an
oxidizing synthesis. We are, therefore, still unable to decide the exact
manner in which urea is produced.

We also desire to call attention to a compound present in urine, although
only in small amount, which has often been mentioned in relation to urea.
This is creatinine:

NH — CO
/
C= NH
\

N(CH3) CHa

The amount excreted daily by human beings varies from 0.6 to 1.3
gram. It is the anhydride of creatine, which is present in the muscles,
and is a methyl-guanidine-acetic acid:

NHa
/
C = NH
\

NCCHa) .CH2.COOH.

On boiling with baryta water creatine decomposes, adding water, and
forms urea, sarcosin, and other products. It may also be obtained syn-
thetically by heating sarcosine (methyl-glycocoll) with cyanamide in a
sealed tube to 100 degi'ees,' or by adding a few drops of ammonia in the
cold to a saturated solution of sarcosine containing the equivalent amount
of cyanamide, and allowing the mixture to stand: ^
NH2 CH3 NH2

I I I

CN + N— H = C = NH

CH2 . COOH NCCHa) .CHg.COOH.

Cyanamide Sarcosine Creatine

Creatine may also be considered a substituted guanidin, as the following

formula shows: ^^_

NH2

I

C = NH

I

NH2
Guanidine

J. Volhard: Zeit. f. Chem. 1869, .318.
A Strecker: Jsb. Chem. 1868, 686.

236 LECTURE XL

We have already indicated, while discussing arginine and arginase, that
urea can be formed from guanidine. Creatine, therefore, may also be
looked upon as an antecedent of urea, although we do not possess any
confirmatory proof of this. The position of creatine in the general
metabolism is, in fact, extremely uncertain. We do not even know its
source. It is generally considered as being directly related to the proteins.
Its formation from albumin has, as yet, never been proven. It appears
far more probable that it is more directly related to the nourishment; at
any rate, the elimination of creatine depends upon the introduction of
food. It is principally found in the muscles, although it has also been
isolated from the blood, brain, transudates, and in the amniotic fluid.
It is stated that the amount of creatine is increased by muscular work.
This has given rise to the assumption that its formation is related to
muscular contraction. It is, however, also possible that its elimination
may be due to an increased circulation of the blood and the changes which
occur in a working muscle.

Uric acid must also be looked upon as a source of urea. It has been
shown that a part, at least, of this substance when administered to the
organism of mammals, is changed into urea.' This form of urea produc-
tion plays only an insignificant part in mammals. It was long thought
that uric acid was first formed from albumin, and that this was then con-
verted into urea. It was believed to have been observed that a dimin-
ished oxidation in the tissues was responsible for an increase in the
amount of uric acid at the expense of urea. Such an assumption was
supported by th'e observation that those animals, namely the reptiles,
whose metabolism is very slow, showed only uric acid in their excreta.
Opposed to this assumption was the fact that birds, whose metabolism
we generally consider to be a very rapid one, likewise e.xcreted the larger
part of the nitrogen from their food in the form of uric acid. To-day,
the question of uric acid production in the organism of mammals, and
also the other vertebrates, has been more satisfactorily explained. It is
quite independent from that of urea. We shall see later, that the uric
acid of mammals, in contradistinction to that of birds and reptiles, has
no relation whatever to alljumin-metabolism, but is derived from the
nucleins. We shall consider this in connection with the constitution of
uric acid and related compounds, and shall deal with the subject here only
in so far as is related to the metabolism of albumin.

There are, undoubtedly, direct relations between the decomposition of
albumin and the elimination of uric acid in those animals in which the
larger part of the nitrogen administered reappears in the form of uric
acid. This is especially noticeable with reptiles. Here the elimination of
uric acid stands in the same relation to the albumin taken into the

» Wohler and Frerichs: Ann. 65, 335 (1S48); Neubauer: Ann. 99, 206 (1856).

ALBUMINS OR PROTEINS. 237

system, as does urea in the case of mammals. The analogy between uric
acid and urea has received further support from the discovery that the
same materials which produce an increase in urea in mammals, will cause
an increased elimination of uric acid in birds. Thus, von Knieriem ' showed
such an influence by the administration of amino acids, while von Schroder -
observed the same thing by feeding ammonium salts. Urea,' also, will
cause an increased elimination of uric acid.

Minkowski * obtained a further insight into the production of uric acid
in birds, by extirpating the liver of geese. These animals will survive
the operation for 20 hours, because, in them, the portal vein is not the
only outlet of the splanchnic vessels, but is supplemented by another,
the Vena communicans. The elimination of nitrogen during this opera-
tion was somewhat diminished. The greatest change occurred in the
uric acid of the urine. Normal geese eliminate from 60-70 per cent of
the total nitrogen in the urine as uric acid, while those whose livers had
been removed showed only 3.6 per cent. Ammonia, in large amounts,
took the place of uric acid. Minkowski also noticed at the same time a
large amount of sarcolactic acid in the urine, a substance which is en-
tirely absent in the urine of normal geese. The question now arises, What
relation do the eliminated ammonia and lactic acid bear to the formation
of uric acid?

We can imagine that the increased formation of ammonia arises from
a secondary process due to the appearance of the lactic acid. We are
aware of the fact that in ammonia the animal organism has a valuable
weapon to protect itself against acids. An increased appearance of
ammonia in the urine is very often a direct indication of an increased
production of acid in the organism. That the presence of the lactic acid
was an important factor in the increased production of ammonia is evident
from the fact that an addition of alkali caused an appreciable diminution
of the amount of ammonia in the urine.' At any rate, a part of the am-
monia must be looked upon as exerting a neutralizing effect.

Hoppe-Seyler " has shown that the presence of the lactic acid was due
to a diminished oxidation in the tissues, as a result of the operation. It
is well known that a lactic acid formation often accompanies a dimin-
ished supply of oxygen. Minkowski ' has replied to this objection by
showing that the total extirpation of the liver is not the only way an

' Z. Biol. 13, 36 (1877).

' Z. physiol. Chem. 2, 228 (1878).

' H. Meyer: Inaug. Diss. Konigsberg, 1877.

' Arch., exp. Path. Pharm. 21, 89 (1886); 31, 214 (1893).

' S. Lang: Z. physiol. Chem. 32, 320 (1901).

° Festschrift zu Vichow's 70 Geburtstag, 1891. Cf. also Araki: he. cil.

' Arch. exp. Path. Pharm. 31, 214 (1893).

238 LECTURE XI.

excretion of lactic acid can be caused, but that even ligating the vessels
of the Hver is sufficient to bring this about. Lactic acid will only appear
in the urine at the moment when the last branch of the hepatic artery has
been tied. There is no lactic acid formed, if a single branch is left free.

Kowalewski and Salaskin ' have shown that the appearance of lactic
acid after the extirpation of the liver is actually to be traced back to a
disturbance in the formq,tion of uric acid. They could detect the forma-
tion of lactic acid by merely leading ammonium-lactate through the liver
of birds. H. Wiener'' also showed relations between the lactic acid and
the formation of uric acid. He fed lactic acid and urea to birds, and
noticed an increase in the uric acid.

In order to give an idea of the relationship of lactic acid to uric acid
it will be necessary to give the constitution of uric acid. It is a 2, 6, 8
trioxypurine : HN— C = O

I I
0 = C C— NH

I I >co

HN— 0— NH

Uric acid

Horbaczewski has obtained it syntheticallj- by heating urea and glycocoll
together, and also by heating trichlorlactamide with an excess of urea.
Uric acid is decomposed on strongly heating into urea, hydrocyanic acid,
cyanuric acid, and ammonia. If uric acid is heated with concentrated
sulphuric acid in a sealed tube at 170° C. it breaks up into glycocoll, carbon
dioxide, and ammonia. Strecker,' to whom we owe this discovery, com-
pares the uric acid production from the components glycocoll and cyanic
acid with the production of hippuric acid from glycocoll and benzoic acid.

We observe from this that the .synthesis of uric acid from lactic acid
and ammonia, or urea, is a very plausible one. The standpoint that
uric acid is a direct degradation product of albumin has long been dis-
carded. Uric acid, which obtains its nitrogen from albumin, can only be
produced synthetically. We wish to call attention at this point to the
very close analogy between the production of uric acid and that of urea.
It can be shown in both cases that ammonia, directly or indirectly, plays
a part, as does also an acid (carbonic acid or lactic acid). The ammonia
in both cases may have the same origin, being derived from albumin or its
cleavage-products. The organisms of the birds and reptiles evidently are
also capal^le of causing the removal of the NH2 group. This is appar-

' Z. physiol, Chem. 33, 210 (1901).

' Verh. XVII, Kong. Med. 1889, p. 622, and Arch. exp. Path. Pharm. 42, 375
(1899); Verh. XIX, Kong. Med. 1901, 383. Hofmeister's Beit. 2, 42 (1902). Cf. also
H. Wiener: Die Hamsaure, Ergebnisse der Physiologie (Asher and Spiro) 1, I, 555
(1902).

' Ann. 146, 142 (1868).

ALBUMINS OR PROTEINS. 239

ent from the comparatively large production of lactic acid. The latter

may be derived from various sources. Here, the carbohydrates, as well as

the amino acids, come into consideration. We know some of these which

are closely related to lactic acid. We would refer especially to alanine :

CH3 CH3 CH2.OH CHo.SH

III I

CH.NH2 CHOH CH.NH2 CH.NH2

COOH COOH COOH COOH

Alanine Lactic Acid Serine Cysteine

We can also imagine that serine and cysteine may bear some relation to
the formation of lactic acid. Leucine might also produce lactic acid, if we
assume that its carbon chain is broken in the middle:
CH3 .CH3

CH2

CH .NHo » Alanine

COOH

Leucine

We can, therefore, easily derive all of the components of uric acid
direct from albumin. It is not to be implied, however, that lactic acid
may not also arise from other sources.

It might be thought that some idea of the formation of urea could be
obtained from the way uric acid is produced. We, however, know that
there are several ways in which the uric acid formation may be explained.
We can take all of our theories for the formation of urea and apply them
directly to that of uric acid. It is possible that the synthesis in this case
is primarily carried out with the elimination of water; although it is also
conceivaltle that an oxidation synthesis may be a factor. It is not at all
impossible that the formation of urea may play a part in the synthesis of
uric acid.

Wiener's investigations will give us an idea of these relations. He

COOH
CHOH
COOH

NH— CO

I I

CO CHOH

I I

NH— CO

found that tartronic acid:

and its ureide (dialuric acid) :

240 LECTURE XI.

easily go over into uric acid; in fact, when isolated organs are used, this
result has been accomplished. Wiener assumes from this that lactic acid:

CH3

CHOH

I
COOH

goes over into tartronic acid:

COOH

CHOH

I

COOH
which then forms dialuric acid:

NH— CO

I I

CO CHOH

I I

NH— CO

and finally, by the addition of the urea radical, produces uric acid:

NH— CO

I I

CO C— NH

I II >co

NH— C— NH

We should, therefore, have to assume that the main cause of the disturb-
ance in the synthesis of uric acid, after the liver had been removed, was
the non-oxidation of lactic acid into tartronic acid, and, evidently, also, the
non-formation of urea.

It is clear that we are not yet prepared to state that the formation
of uric acid proceeds normally in this manner. Wiener's investigations
at all events indicate the manner in which the synthesis may proceed.
Wiener also advances the opinion that the organisms of human beings
and mammals in general synthesize at least a part of their uric acid, and
he sees no real difference in principle between their metabolism of albumin
and especially in the formation of their end-products from that of birds
and reptiles. The distinction is rather a quantitative one. Birds and
reptiles likewise produce urea, but the amount formed is less than that of
uric acid. On the other hand, urea predominates in human beings and
mammalia. It cannot be denied that such an assumption has much to
commend it. Nowhere in the animal kingdom do we observe any sharp
demarcations, especially in those processes which are of such great impor-
tance as is the case with metabolism. The synthetic formation of uric
acid among mammals, according to our present knowledge, must, never-

ALBUMINS OR PROTEINS. 241

theless, be relegated to the background. Even if such a process does
take place, the amount produced thereby is so small in comparison with
that obtained from other sources, that it possesses little significance.
Wiener has also tried to show that uric acid is synthetically produced by
mammals. His conclusions have, however, been contradicted.' The
synthetic production of uric acid by the mammalian organism cannot, at
present, be accepted with certainty.

The question now arises, In what organs does the uric acid formation
take place? The liver seems to play an important part in this process
among birds. W. v. Schroder ^ extirpated the kidneys from hens, and
succeeded in keeping these birds alive for 5-10 hours. Schroder examined
after their death the blood and organs for uric acid, and found that a very
appreciable accumulation of this acid had resulted. The uric acid pro-
duction had evidently continued after the extirpation of the kidneys.
The same behavior was noticed with snakes. It seems, therefore, that
the kidneys are of little, or even no, service in this process. This de-
cision was the more striking, as the kidneys were for a long time looked
upon as the most important place for the production of uric acid. This
opinion had originated from the way uric acid is distributed after the
ureter has been ligated.

Both of the albumin metabolic substances so far considered are con-
nected with the whole albumin molecule and all its cleavage-products.
Ammonia ought also to be included in this group, as it occurs in varying
quantities in urine. It was formerly believed that an increased elim-
ination of ammonia indicated an insufficient production of urea. Little
by little the cause was more accurately investigated, and it was dis-
covered that the increase in the amount of ammonia was not a primary
effect, but that it was due to an increased production of acid. Thus, in
a diabetic, the appearance of acetoacetic acid and of /?-hydroxy-butyric
acid is associated with an increased elimination of ammonia. F. Walter '
even showed that the administration of hydrochloric acid to human
beings and dogs caused an increased elimination of ammonia. A.
Schittenhelm and A. Katzenstein * have recently shown that the amount
of ammonia present in the urine is directly related to the total nitrogen of
the urine. It rises and falls with the consumption of albumin, so that the
ratio of ammonia to the total nitrogen remains constant within narrow
limits. The amount of ammonia excreted is not affected by the admin-
istration of urea or ammonium carbonate. It is also interesting to note
that, not only does an increased ehmination of ammonia follow an increased

' Cf. R. Burian: Z. physiol. Chem. 43, 497 (1905).
' Arch. Anat. Physiol. 1880, p. li:^, Supplement.
' Loc. cil.
* Arch. exp. Path. Therapie, 2, 541 (1905).

242 LECTURE XL

diet of albuminous material, but that this also occurs after the addition
of the free amino acids, glj-cocoll and alanine. It is very significant that
the administration of ammonium carbonate is followed by a sharp decline
in the formation of ammonia, so that the ratio of ammonia-nitrogen to
the total nitrogen in the urine becomes less than under normal conditions.
We cannot conceive that the amino acids mentioned are capable of pro-
ducing acidosis on their own account, although we can imagine that
Siegfried's discovery, that the amino acids take on carbon dioxide forming
carbamic acids, is here expressed. On the other hand, this intermediate
acidosis gives us an indication of the manner in which the decomposition of
the albumin cleavage-products is carried out in the tissues. It does not
seem improbable that acids are temporarily formed after the ammonia
has been split off, which cause an increase in the production of ammonia.
Our knowledge of the intermediate metabolism of albumin is so slight
at present that we have hardly any conception of these relations.

We will now consider the end-products of albumin metabolism, which
occupy a different position from those just mentioned in that they are
not derived from the albumin cleavage-products as a whole, but can be
traced to definite amino acids. Among these is hippuric acid. It was
discovered in horse urine by Liebig. Its method of formation was known
to Keller and Wohler.' These authors noticed that when benzoic acid
was administered per os, it did not reappear as such in the urine, nor
could cleavage-products be found which were related to it. Keller and
Wohler, however, noticed an appreciable increase in the amount of hip-
puric acid. Its manner of formation is evident from its constitution. It
is broken down on boiling with strong mineral acids or alkalies, with the
addition of water, into benzoic acid and glycocoll:

CeHs.CO.NH.CHo.COOH + H2O = CgHs.COOH + NHo.CHo.COOH
Hippuric acid Benzoic acid Glycocoll

It may be synthetically produced from benzamide and monochlor-
acetic acid:

C6H5.CONH2 -f Cl.CHo.COOH = CsHs.CO.NH.CHa.COOH + HCl

Benzamide Monochloracetic Hippuric acid

acid

It may also be obtained by heating glycocoll with benzoic acid in a
sealed tube for 1 or 2 hours at 160 degrees.

The observation of Keller and Wohler, that benzoic acid appears in the
urine united to glycocoll, has been confirmed by numerous investigations

' Ann. 43, 108 (1842); Wohler and Frerichs; 65, 335 (1848); Wilhelra Wiechowski;
Hofmeister's Beit. 7, 204 (1905).

ALBUMINS OR PROTEINS. 243

through administrations by the mouth, as well as by subcutaneous injec-
tions. We know to-day that this synthesis, which at that time created
much excitement on account of the fact that it was the first instance in
which a synthetic process had been shown to take place in the animal
organism, is not unique. Thus, R. Cohn ' found that naphthoic acid
administered to rabbits and to dogs reappeared in the urine as naphturic
acid. Its formation is exactly analogous to that of hippuric acid:

CioHy.COOH + NH2.CH2.COOH = C10H7.CO.NH.CH2.COOH + H2O
Naphthoic acid Glycocoll Naphturic acid

Salicylic acid unites with gl3-cocoll in the same manner.^ Hydroxy-
hippuric acid is formed:

OH.C6H4.COOH + H2N.CH2.COOH = OH.C6H4.CO.NH.CH2.COOH

Salicylic acid Glycocoll Hydroxyhippuric acid

+ H2O

It is also interesting to note that alkylated benzoic acids, for instance,
toluic acid,3 likewise unite with glycocoll, reappearing as alkylated hippuric
acids:

CH3.C6H4.COOH + NH2.CH2.COOH = CH3.C6H4.CO.NH.CH2.COOH

Toluic acid Glycocoll Toluric acid

+ H2O

Phenylacetic acid similarly appears in the urine as phenaceturic acid:^

C6Hs.CH2.COOH + NH2.CH2.COOH = C6H5.CH2.CO.NH.CH2.COOH

Phenylacetic acid Glycocoll Phenaceturic acid

+ H2O

We have already seen, when discussing glucuronic acid, which plays a
role in the animal organism very analogous to that of glycocoll, that the
cells are able to adapt compounds, which of themselves would not unite
together, partly by oxidation, partly by reduction, and sometimes by both
methods. Thus, toluene is first converted into benzoic acid and then
united to glycocoll. Ethyl- and propylbenzenes are changed in the same
manner.6 Xylene is Hkewise oxidized to toluic acid. It is interesting to
note that aldehydes are oxidized to acids; as an example we will cite

' Z. physiol. Chem. 18, 112 and 119 (1894).

' Bertagnini: L. Ann. 97, 248 (1856); Z. physiol. Chem. 1, 244 and 253 (1877-78).

^ Schultzen and Naunyn: Du Bois' .'^rch. 1867, 352.

* E. and H. Salkowski: Z. physiol. Chem. 7, 161 (1882-83); 9, 229 (1885).

^ M. Nencki and P. Giacosa: ibid. 4, .325 (1880).

244 LECTURE XI.

the conversion of nitrobenzaldehyde into nitrobenzoic acid, and the sub-
sequent formation of nitrohippuric acid:'

NO2 . C6H4 . CHO + O = NO2 . C6H4 . COOH
Nitroljenzaldehyde Nitrobenzoic acid

NO2.C6H4.COOH + NH2.CH0.COOH = NO2.C6H4.CO.NH.CH2.COOH

Nitrobenzoic acid Glycocoll Nitrohippuric acid

+ H2O

The conversion of benzamide ' into hippurie acid is especially note-
worthy on account of the fact that water must be added to form benzoic

^'^^^' C6H4.CONH2 + HoO = C6H4.COOH + NH3

Benzamide Benzoic acid

The ability of the organism to unite other substances with glycocoll
is not confined to benzoic acid or its derivatives, but the same is true of
the carboxylic acids of furan-, thiophen- and pyridine-nuclei. Thus, from
thiophenaldehyde ^ thiophenic acid is formed, which in the presence of
glycocoll goes over into thiophenuric acid:

C4H3S . CHO + 0 = C4H3S . COOH

Thiophenaldehyde Thiophenic acid

C4H3S.COOH + NH2.CH2.c6OH = C4H3S.CO.NH.CH2.COOH + H2O

' Thiophenuric acid

Such reactions in the animal organism are of interest in more than one
way. Such investigations give an idea of the activity of the animal cell.
We notice that oxidation and reduction processes are carried out with the
greatest ease, while water is split off, or added, as the case demands.

We ask ourselves, Where does the animal organism obtain the glycocoll
which enters into these combinations? We have seen that this amino
acid is found among the many cleavage-products of the albumins. There
can be no doubt that the animal cells obtain the necessary glycocoll
by the decomposition of proteins. The fact that glycocoll will unite with
benzoic acid and analogous compounds under technical conditions, per-
mits us to draw valuable conclusions regarding the decomposition of
albumins in the tissues. There is little doubt that this also applies to
the amino acids. The glycocoll formed is usually further disintegrated
into urea; if, however, benzoic acid happens to be present in the tissues,
the glycocoll, produced as an intermediate substance, is removed from
further metabolism. We have already seen that other albumin cleavage-

' Ibid. 17, 274 and 292 (189.3).

' L. V. Nencki: Arch. exp. Path. Pharm. 1, 420 (1873).

» R. Cohn: Z. physiol. Chem. 17, 281 (1893). Cf. E. Fromm: Die chemisehen
Schutzmittel d. Tierkorpers bei Vergiftungen, K. J. Triibner, Strassburg, 1903, p. 14;
also M. Nencki: Opera omnia (Vieweg and Sohn, Braunschweig, 1905).

ALBUMINS OR PROTEINS.

245

products can be combined in like manner. Thus, on administering ben-
zoic acid to birds we obtain a definite amount of ornithine. In this case
hippuric acid does not appear in the urine, being replaced by ornithuric
acid, the dibenzoyl compound of ornithine. As previously stated, we can
unite cystine with brombenzene, in a dog, thus protecting it from further
oxidation. All of these discoveries indicate that the disintegration of
albumin in the tissues proceeds in an analogous manner to that of the
fats or carbohydrates. Glycogen is first split up into its components and
then consumed. The same kind of action takes place with the fats.

We might ask ourselves whether the glycocoll withdrawn from the body
by the benzoic acid administered can be directly derived from the amount
of decomposed albumin. An answer to this question must be obtained
by studying the increased elimination of hippuric acid caused by the
administration of benzoic acid, and tracing at the same time the dis-
integration of the albumin. Such experiments have been made,' and it
appears that more glycocoll is excreted than can be derived from the dis-
integrated albuminous substance. Such results are to be very cautiously
accepted, as we know comparatively little about the decomposed albu-
mins in the intermediate metabolism. We must always consider the
possibility that the animal cell may be capable of producing glycocoll from
other amino acids. We have already called attention to a striking example
of this by showing that it was impossible to change the composition of the
individual amino acids in the serum-albumins bj' feeding a protein which
was especially rich in a certain amino acid.^ ' In the case indicated, the
" normally " constituted serum-albuminous bodies were evidently produced
from gliadin. A glance at the following table will show the changes
which must have taken place. We find it necessary to state, in order to
prevent any misunderstanding, that our conclusions were naturally only
superficial and incomplete. The configuration and stereochemistry will
at some time undoubtedly be taken into consideration in studying such
changes.

100 parts of Albumin contaia

albumin.

Gliadin.

3.5

0.7

2.2

2.7

present

0.33

18.7

6.0

2.8

2.4

3.8

2.6

8.5

31.5

2.5

1.3

2.5

2.4

present

present

Glycocoll . .

ALanine

Aminovaleric acid

Leucine

Proline

Phenylalanine . .
Glutamic acid . .
Aspartic acid . .

Tyrosine

Tryptophane . .

> Cf. W. Wiechowski: Arch. exp. Path. Pharm. 63, 435 (1905).
' E. Abderhalden and F. Samuely: loc. cit.

246 LECTURE XI.

The question arises, What has become of the large amount of glutamic
acid? It is possible that it was completely disintegrated in the intes-
tine. This phenomenon may perhaps indicate the reason for the marked
increase in the amount of ammonia in the blood of the portal vein during
digestion. We might, however, assume that the glutamic acid was con-
verted into other amino acids and utilized in the synthesis of albumin.
We are unable to form a definite opinion. It is important, however, to
point out the possibility of such transformations, because the conclusion
has been drawn from the considerable amount of glycocoll which can be
withdrawn from the organism by means of benzoic acid, that the breaking
down of all the amino acids to urea passes through the glycocoll stage.'
We are not justified in making any such assumption. There is no founda-
tion for it. It is far more probable that the organism in any given case
utilizes its albuminous substance richest in glycocoll, or, in case of neces-
sity, forms glycocoll from other amino acids. It must not be forgotten
that benzoic acid is a poison to the cells, causing an increase in the dis-
integration of albumin, thus leading to a direct increase in the amount
of glycocoll. We must also notice another possible source of glycocoll.
We shall soon see that the animal organism is able, to a marked degree, to
decompose uric acid. It is assumed, to be sure without an entirely satis-
factory proof, that glycocoll is formed as a decomposition product. The
'amount of this amino acid thus formed is necessarily small among
mammals. Finally, we must consider the possibility of glycocoll being
produced synthetically, for instance, from ammonia and acetic acid. We
have, to be sure, not yet succeeded in detecting such a synthesis.^

All of the benzoic acid administered is not changed into hippuric acid.
A part is excreted as such, and another portion cannot be traced, evidently
being transformed in some unknown manner.

The next question is. In which organ is the hippuric acid produced?
G. V. Bunge and 0. Schmiedeberg ^ studied first of all the livers of frogs.
These survive the extirpation of the liver very well indeed, and live for
3 or 4 days after the operation. They formed hippuric acid when benzoic
acid was introduced into the dorsal lymph sac, and particularly large
amounts when glycocoll was added at the same time. Hippuric acid has
never been obtained from the organisms of frogs or their excretions with-
out the previous administration of benzoic acid. The liver of the frog is,
therefore, not the only organ in which the benzoic acid unites with glycocoll,
if, indeed, the liver participates at all in this synthesis.

Bunge and Schmiedeberg next tested the kidneys to see if they were
able to produce hippuric acid from benzoic acid and glycocoll. They

' W. Wiechowski: loc. cil.

2 R. Oohn: Arch. exp. Path. Pharm. 53, 4.35 (1905).

3 Arch. exp. Path. Pharm. 6, 233 (1S77).

ALBUMINS OR PROTEINS. 247

ligated the vessels of the kidneys of dogs, and then introduced benzoic
acid and glycocoll into the remaining circulation. The animals experi-
mented upon were killed after 3 or 4 hours, and the blood and liver
tested for hippuric acid. Benzoic acid, but never hippuric acid, was
found. The exact proof that the kidneys of the dog is as a matter of
fact the place where the synthesis of hippuric acid is effected, Bunge
and Schmiedeberg determined by direct experiment. They cut out the
kidneys from a dog that had been just killed, and passed defibrinated
blood, to which benzoic acid and glycocoll had been added, through the
renal arteries. It flowed away through the veins of the kidneys and
returned through the arteries, this process being continued for several
hours. Hippuric acid was then found in this blood as well as in the
fluid which flowed from the ureter. The other kidney and a part of
the original blood were used as a control, but no hippuric acid was
found in them. The surviving kidney had, therefore, produced hippuric
acid from glycocoll and benzoic acid. When the experimenters added
only benzoic acid, but no glycocoll, they found that the amount of
hippuric acid formed was very small. This, however, quickly increased
when glycocoll wa.s added and passed through the kidney. The syn-
thesis v/as just as satisfactory at the room-temperature as it was at
37° C.

The red blood-corpuscles and the cells oi the kidneys are of great impor-
tance in the synthesis of hippuric acid. When the kidney tissues are
destroyed by chopping, or, better yet, by rubbing them up with pulverized
glass, we find that the conjugation of glycocoll with benzoic acid no longer
takes place. When the kidneys are cooled to — 20 degrees and then raised
to 40 degrees, it is also found that hippuric acid is no longer produced
from its components. Again, the synthesis could not be effected if the
serum of the blood, instead of the blood itself, was utilized. It has been
shown, by the investigations of A. Hoffmann, that oxygen plays an impor-
tant part in this synthesis.' He led blood through the kidneys, in which
the oxygen had been displaced by carbon dioxide, and foimd that no syn-
thesis of hippuric acid resulted. Quinine also prevented the kidney cells
from producing hippuric acid.

It-seems very probable that the synthesis of hippuric acid from glycocoll
and benzoic acid is due to a ferment, water being split off. The attempt
has been made to isolate such a ferment. Recent investigations in
which, contrary to earlier experiments, it was found possible to detect
the synthesis in the chopped up kidneys, lead to the hope- that the

' A. Hoffmann: Arch. exp. Path. Pharm. 7, 233 (1S77).

' W. Kochs: Pfliiger's Arch. 20, 64 (1879). M. R. Berminzone: Bol. accad. med. di
Genua 16, No. 1 (1901). J. E. Abelous and H. Ribaut: Compt. rend. Soc. Biol. June 9,
1900.

248 LECTURE XI.

conjugation of glycocoll with benzoic acid can be accomplished without
the direct use of organs or cells.

As regards the place where the hippuric acid is formed, it is to be noted
that what has been said applies only to dogs. Frogs produce hippuric
acid even after the extirpation of the kidneys. Salomon ' also observed
hippuric acid in large amount after the administration of benzoic acid to
a rabbit whose kidneys had been removed. It is poissible that the syn-
thesis of hippuric acid is more localized in the carnivora than it is with the
herbivora, because the formation of hippuric acid by the former under
normal conditions is only very small in amount. The quantity of hippuric
acid daily excreted by human beings under an ordinary diet is about 0.7
gram. It may be increased to more than 2 grams by a liberal diet of
vegetables or fruit.

Glycocoll not only participates in the production of hippuric acid and
of the other artificially introduced products just mentioned, but is also a
component of glycocholic acid and gh/cocholeic acid. Both are decom-
posed, in the same manner as is hippuric acid, by boiling with acids or
alkalies into glycocoll, cholic acid, or choleic acid, respectively. These
last two acids are both found as constituents of the bile.

Besides these, there is another acid containing sulphur called tauro-
cholic acid which is found in the bile of most animals, and is likewise
related to one of the albumin ■cleavage-products; i.e. to cystine. When
taurocholic acid is heated with acids or alkalies, it is decomposed into
taurine and cholic acid. The relations of taurine, which is an amino-ethyl-
sulphonic acid, to cystine and cysteine, are evident from the following
formula? :

CHaSH CH, . SOoOH CH.> . SO., . OH

CH . NHo CH . NH2 CH9 . NH2

I I

COOH COOH

Cysteine Cysteinic acid Taurine

Friedmann has succeeded, as previously mentioned, in converting
cysteine into cysteinic acid, and this into taurine. Shortly after the chemi-
cal relations between these compounds had been settled, experiments with
animals also indicated the probable derivation of taurine from cysteine.
W. V. Bergmann^ fed dogs, in which he had made a complete biliary
fistula, with cysteine, and estimated the amount of taurocholic acid
separating out with the bile. He could not detect any increase in the
sulphur content of the bile in these experiments, but did notice such

' Z. physiol. Chem. 3, .365 (1879).
' Hofmeister's Beitr. 4, 132 (1903).

ALBUMINS OR PROTEINS. 249

when sodium chelate, that is, the other component of taurocholic acid,
was added at the same time with the cysteine. Wohlgemuth ' confirmed
these experiments, and showed with rabbits that the amount of sulphur
in the bile and the sulphur content of the hver increased with the admin-
istration of cysteine alone.

We know nothing definite concerning the further history of tauro-
cholic acid or of the taurine contained in its molecule. Salkowsld ^ found
that, after the administration of taurine to human beings and dogs, a
part of this taurine and a substituted urea,

/NH2
C=0

^ ^ CH2— CH2 . SO3H
appeared in the urine.

Almost all of the sulphur of the taurine reappears in the urine as sul-
phuric and sulphurous acids, when fed to a rabbit.

Salkowski could not detect any increased elimination of sulphuric acid
nor of sulphurous acid, after taurine had been fed to human beings
and dogs. , Cysteine increases the elimination of sulphuric acid in the
urine of human beings and dogs.' The same also is observed in the case
of rabbits, while here salts of hyposulphurous acid are found in addition.
We will add that thiosulphuric acid has been found in the urine of cats
and dogs.

It has been shown recently * that cystine, in the form of polypeptides, is
apparently decomposed during metabolism in the same manner as when
cystine itself is administered. It is interesting to note that in these experi-
ments there was a distinct increase in the o.xidized sulphur of the urine
corresponding to the duration of the experiment. The urine always
contains a part of the sulphur in an unoxidized form. This portion is
also called " neutral " sulphur. Its amount varies and to some extent is
directly related to the oxidized sulphur.

It is at present impossible to give a clear outline of the relations of the
components containing sulphur of the urine to albumin or its cleavage-
products, because we are not yet able to recognize all the constituents of
proteins which contain sulphur. We can only consider the fact as settled
that the sulphur in the cystine administered to an animal organism largely
reappears in an oxidized form in the urine; in fact, as sulphuric acid.
There is no doubt but that cystine is also formed during a normal disinte-

' Z. physiol. Chem. 40, 81 (190.3).
' Virchow's Arch. 58, 460 (1873).

' Cf. E. Goldmann; Z. physiol. Chem. 9, 260 (1885). C. H. Rothera: J. Physiology,
32, 17.5 (190.5V L, Blum: Hofmeister's Beit. 5, 1 (1903).

« E. Abderhalden and F. Samuely: Z. physiol. Chem. 46, 187 (1905).

250 LECTURE XI.

gration of the albumins, and that this is decomposed in the same manner
as the cystine which is artificially introduced.

The sulphuric acid of the urine has been subjected to thorough study.
It was found that it occurred in various combinations. If we add barium
chloride to urine which has been previously acidified, barium sulphate will
precipitate at once. A further turbidity will appear after filtering this
off and boiling with hydrochloric acid. E. Raumann ' satisfactorily
explained this behavior of sulphuric acid in urine. The sulphuric acid
at first precipitated is derived from sulphates — salts of sulphuric acid.
That which is obtained after boiling with hydrochloric acid is due to
sulphuric acid which has been in combination with different aromatic
sul)stances in the urine. The hydrochloric acid decomposes these aromatic
compounds — also called sulphuric acid esters — into the aromatic com-
ponent and sulphuric acid, the latter being then precipitated by barium
chloride. The sulphuric acid esters themselves form soluble barium salts.
We shall see that sulphuric acid forms the same kinds of compounds with
the.se that we found it does with glycocoll and glucuronic acid. We wish
to add at this point that some sulphur compounds still remain in solution
even after the sulphur in the sulphuric acid esters have been precipitated.
This is the "neutral sulphur" mentioned above. In order to detect this
sulphur it is necessary to oxidize it, thus converting it into sulphuric acid,
when it will be precipitated by barium chloride. By these methods we
are able to isolate all three varieties of combined sulphur in the presence
of one another. The determination of the total sulphur together with
that of the total nitrogen in the urine gives us a very good conception of
the course of the disintegration of albumin.

We must not forget to mention that sulphocyanic acid, or thiocyanic
acid, HCNS, also occurs in urine. Gscheidlen ' found it invariably present
in the urine of human beings, horses, calves, dogs, cats, and rabbits. In
human urine 0.2 to 0.8 gram is eliminated daily. The sulphocyanic acid
comes from the saliva, being formed in the salivary glands. This acid
reaches the blood-stream by absorption. If all the ducts of the salivary
glands are cut and the saliva discharged externally, sulphocyanic acid no
longer appears in the urine. Its origin and significance have never been
exDlained.

' Ber. 9, .54 (1876).

' Tageblatt 47, Versammlung deutscher Naturi. u. Aerzte in Breslau, 1874.

LECTURE XII.

ALBUMINS OR PROTEINS.
VI.

Metabolic End-Products.

We have already mentioned the fact that putrefactive processes always
take place in the intestines to a greater or less extent. A part of the
products thus formed is absorbed and eliminated in the urine. Only a
small proportion of these compounds is excreted unchanged, or combined
with alkalies. By far the largest percentage of the albuminous cleavage-
products produced by putrefaction appear in the urine in the form of com-
plex combinations. Baumann ' and Brieger - have shown that here the
acid esters of sulphuric acid are most important. The organism produces
glucuronic acid only when there is a deficiency of sulphuric acid. There
always seems to be in the urine a small amount of conjugated glucuronic
acid compounds. The decomposition products are largely the aromatic
components of albumin; in fact, chiefly tyrosine and probably phenyl-
alanine as well. Tryptophane is likewise an important factor. We have
seen that tyrosine, which is p-hydroxyphenyl-«-aminopropionic acid, is
changed by loss of ammonia into p-hydroxyphenylpropionic acid; the
latter on further oxidation and loss of carbon dioxide finally decomposes
into p-cresol and phenol. From tryptophane skatole and indole are the
end-products obtained.

The most important of the conjugated sulphuric acid compounds are
phenyl-, tolyl-, indoxyl-, and skatoxyl-sulphuric acids. Catechoyl-sul-
phuric acid is also, although not invariably, found in human urine in
small quantity. We may state that some of the sulphuric acid combina-
tions have not yet been identified. The amounts of such substances in
horse urine are especially large. In human urine, on the other hand,
there is much le.ss present than of the other sulphur compounds. From
0.1-0.6 gram is excreted on an average every 24 hours. Experience has
shown that no definite values can be given. They vary considerably, and
are naturally dependent on the putrefactive changes in the intestines.
The excretion of the acid esters of sulphuric acid can be increased artifi-

' E. Baumann: Ber. 9, 54 (1876); 9, 1389 (1876); 9, 1715 (1876); 10, 685 (1877); 11,
1907 (1878); 12, 2166 (1879); Pfiuger's Arch. 12, 63 (1876); 12, 69 (1876); 13, 285
(1876); Z. physiol. Chem. 1, 60 (1877-78); 2, 335 (1878-79); 3, 250 (1879); 4, 304 (1880);
10, 123 (1886); 17, 511 (1893).

= E. Baumann and L. Brieger: ibid. 3, 254 (1879); 3, 156 (1879).
251

252 LECTURE XII.

daily, for instance, by the administration of phenol. If this be introduced
into the animal organism, it appears in the urine as potassium phenyl-
sulphate.' We assume it to be formed as follows:

CfiHsOH + HO . SO3K = CfiHs . O . SO3K + H2O
Phenol Potassium phenylsulphate

We will add that not only substances like phenol are eliminated in this
way, but in place of phenol we may find: the cresols, CH3 . C6H4OH;
thymol, C3H7(CH3)C6H3 . OH, also the dihydroxy-benzenes, CeH4(OH4)2;
methylquinol, CH3 . 0 . C6H4OH; orcinol, CH3 . C6H3(OH)2; pyrogallol,
C6H3(OH)3; tribromphenol, BrgCeHgOH; o-nitrophenol, NO3.C6H4.OH;
p-amidophenol, NH2 . C6H4 . OH; protocatechuic acid, COOH.C6H3(OH)2;
tannin, salicylamide, m- and p-hydroxybenzoic acids.

Before discussing the sulphuric acid esters which normally occur in
urine, we will rnention the fact that in addition to the above compounds
produced by the artificial introduction of aromatic compounds, substitution
products of the phenols and hydroxyl derivatives of other cyclic com-
pounds may also appear in urine conjugated with sulphuric acid. Such
an example is hydroxyquinolin sulphate, which, to some extent at least,
appears in urine as an acid ester of sulphuric acid. Here also it is inter-
esting to note that in the organism substances are prepared for combina-
tion which of themselves are incapable of reacting together. For example,
benzene is first oxidized to phenol, and then united with sulphuric acid.
We shall soon see that indole and skatole are also first oxidized to indoxyl
and skatoxyl, and then further made to combine:

NH : CgHe -F O = HN : CgHsOH

Indol Indoxyl

HN . CgHs . OH + OH . SO3K = HN : CgHs • O . SO3K-)- HoO

Indoxyl Pot. indoxylsulphate

It is also interesting that substances, themselves capable of combination,
are likewise oxidized. This part of the phenol is oxidized to quinol and
appears then as quinol sulphuric acid:

CsHs . OH + O = HO . C6H4 . OH

Phenol Quinol

HO . C6H4 . OH + HO . SO3K = HO . C6H4 . 0 . SO3K + H2O

Potassium quinol sulphate

These reactions indicate that the formation of sulphuric acid esters is
dependent on the presence of aromatic compounds, whether these are

' E. Baumann and E. Herter: Ber. 9, 1747 (1876); 1, 244 (1877-78).
' C. Brahm: Z. physiol. Chem. 28, 439 (1899).

ALBUMINS OR PROTEINS. 253

artificially administered or normally produced from the food. It is very
probable that, under normal conditions, all the conjugated sulphuric acid
compounds are the outcome of intestinal putrefaction. The amount of
sulphuric acid esters has even been suggested as indicating the extent of
putrefaction taking place in the intestines. We may, however, state that
a true conception of the putrefactive changes in the intestines cannot be
obtained by the determination of sulphuric acid esters alone. Their
amount is naturally dependent, first of all, on that of the absorbed products
of putrefaction, and the absorption depends on the time the material
remains in the intestines. During diarrhea large amounts of putrefactive
products are withdrawn from the organism. The quantity in tiie faeces
would also have to be determined. Moreover, only a part of the putre-
factive products leave the organism in an unaltered condition. If indole
or phenol is administered to the animal organism, it is partially destroyed,
or, more correctly expressed, it cannot be detected in the urine, having
been evidently transformed in some manner.

We must also bear in mind that not all the aromatic putrefactive sub-
stances appear in the urine in the form of conjugated sulphuric acids, but
they are often present as salts, or even in an unaltered condition. We must
also remember that the sulphuric acid present is derived from the albumins
themselves, and is necessarily limited in amount. It is very probable that
larger quantities of glucuronic acid than usual would be obtained in place
of the sulphuric acid esters, if albumins, low in sulphur content, were fed
to the organism. On the other hand, we must admit the possibility of
the organism covering up any such deficit in sulphur by breaking down
proteins rich in sulphur from its own tissues, just as well as our present
knowledge indicates that, within certain limits, the formation of hippuric
acid is independent of the glycocoll in the albumin of the food. As the
amount of stored-up sulphur compounds is necessarily small, it follows
that the animal organism will soon have to rely upon glucuronic acid
when the amount of aromatic putrefactive products exceeds a certain
limit. The compounds conjugated with glucuronic acid are naturally
not detected in determining the amounts of sulphuric acid esters.

Baumann assumed that the acid esters of sulphuric acid were produced
by the combination of aromatic substances with the sulphuric acid residues
which circulated in the body in the form of sulphates. This theory has
recently been questioned. Tauber * only succeeded in obtaining an
increased amount of phenylsulphuric acid in administering large amounts
of phenol only when he introduced sulphites at the same time; while this
was not the case with sulphates. It seems, therefore, that the reaction
between the aromatic compound and the radical containing sulphur

' Tauber: Arch. exp. Path. Pharm. 36, 197 (1895); Z. physiol. Chem. 2, 366

(1878/89); Habilitationsschrift, 1878.

254 LECTURE XII.

occurs before the latter has been oxidized to sulphuric acid. It is very

probable that the final oxidation to sulphuric acid only takes place after

the substances have united. We must not neglect to call attention to the

analogy existing between the formation of the sulphuric acid compounds

and the conjugated glucuronic acids. The latter are also of secondary

nature, only appearing after the dextrose has combined with some of the

conjugating substance.'

We shall now consider those compounds which are produced by the

aromatic cleavage substances of proteins being conjugated with sulphuric

acid. Let us consider first of all phenylsulphuric acid;

CeHfiO . SO2 . OH

and the p-tolyl sulphuric acid:

/CH3

C6H4

^ 0 . SO2 . OH

Both have the same origin and are always classed together. They are
found in urine as alkaU salts. The quantity of each varies, and depends,
as can easily be imagined, on the intensity of the intestinal putrefaction
and the amount of resulting products that is absorbed. Tyrosine is the
mother-substance of the phenols. As we have seen, all the phenol admin-
istered to the body is not excreted as such combined with sulphuric acid
in the urine. A part is changed in another way, probably consumed, while
another portion is in some cases found in the urine, oxidized to quinoyl-
sulphuric acid. This cannot be detected under normal conditions, although
catechoylsulphuric acid is often present in urine, if only in small amount.
Catechol is o-dihydroxybenzene. It has never been definitely decided
whether its sulphuric acid ester is produced by the oxidation of phenol,
or arises from some constituent in the food which is not directly related
to the proteins.

There is some evidence indicating that catechoylsulphuric acid results
from a vegetarian diet, but is not formed in a diet exclusively of meat. It
has been suggested that protocatechuic acid is the mother-substance of
the sulphuric acid ester mentioned.

We will add to the phenyl- and tolylsulphuric acids the other decom-
position products of tyrosine which occur in urine. They may be con-
sidered as intermediate products between tyrosine and phenol. Thus,
there has been found in urine: p-hydroxyphenylpropionic acid (p-hydro-
cumaric acid) : q^j^^ _ qjj ^ ^jj^ _ (.jj^ _ COOH,

and the p-hydroxyphenylacetic acid: '
C6H4 . OH . CH2 . COOH.

' Cf. Lecture II, p. 33.

' E. Baumann: Z. physiol. Chem. 4, 304 (1880); 6, 183 and 234 (1882).

ALBUMINS OR PROTEINS.

255

They have both been obtained from urine, partly as conjugated sulphuric
acids, but mainly in the form of salts.

It is questionable whether these two compounds are always and invari-
ably produced by intestinal putrefaction. It is possible, in fact probable,
that these two hydroxy-acids are produced by the decomposition of tyro-
sine in the tissues. Confirming this is the fact noted by H. Thierfelder
and Nuttal,' that these two acids were observed in the urine of guinea pigs
which had been kept sterile,^ i.e. they had grown up with their intestinal
tracts free from bacteria, consequently there could not lie any putre-
faction. It is important that the urine of these animals did not contain
any typical putrefactive products of proteins; i.e. the phenols.

Para-hydro.xymandelic acid,

CfiHi . OH . CH . (OH) . COOH,

has also been found in the urine, although only under specific conditions.
Schultzen and Ries ^ found it in acute cases of atrophy of the liver.

Blendermann, ^ after feeding tyrosine to a dog, found a dihydroxyphenyl-
propionic acid:

HO . C6H4 . C2H3 (OH) . COOH,
in the urine.

If we review the investigations just mentioned, we shall see that the same
groups of decomposition products are obtained by intestinal putrefaction
of tyrosine as are obtained by the same process outside the animal
organism.^ From tyrosine,

p-hydroxyphenylaminopropionic acid

/OH

C6H4
\

we obtain the following decomposition products:

OH

CH2 . CH (NHo) COOH

p-hydroxyphenylpropionic acid :

p-h}'droxyphenylacetic acid:

/

CeH

\

CHo
OH

p-cresol :
phenol :

/

C6H4

^CH..
/OH

C6H4
s

CHo . COOH

COOH

CH3
Hs . OH

' H. Thierfelder and Nuttal: ihid. 21, 109 (1896); 22, 62 (1897).

= Cf. Lecture IV, p. 64.

' Schultzen and Ries : Ueber akute Phosphorvergif tung u. Leberatrophie, Berlin, 1869.

* H. Blendermann: loc. cit.

• Cf. Lecture VIII, p. 171.

256 LECTURE XII.

We will state once more that according to the experiments at hand,
para-cresol and phenol are to be regarded solely as products of putre-
faction, whereas it is still a question with regard to the other products as
to how much is formed in the intestine and how much is caused by the
intermecUate breaking down of albumin, or of tyrosine, in the cells. It is
unquestionably certain that p-hydroxyphenylpropionic acid and p-hydroxy-
phenyl-acetic acid may be formed beyond the intestine. This discovery
serves to give us an interesting insight into the decomposition of tyrosine
in the tissues. We first observe that the amino group is split off and that
oxidation then sets in. It is still questionable as to how far these observa-
tions may be applied to the decomposition in the metabolism of the cells.
Some observations seem to indicate that the elimination of the amino
group is the first stage of the decomposition of the amino acids.

We must refer to another specific property of tyrosine. In discussing
the digestion of albuminous substances by trypsin, we called attention to
the fact that this amino acid, together with tryptophane, is very quickly
split off from the albumin. It is very possible that this fact may cause
tyrosine and tryptophane, — which we shall soon learn to recognize as the
mother-substance of skatole and indole, — to fall easy prey to the putrefac-
tive bacteria.

Another question of considerable interest confronts us: What becomes of
the other aromatic amino acid, phenylalanine? Investigations on tryptic
digestion show that this amino acid shows an entirely different behavior
from that of tyrosine. It is not set free by trypsin. In the putrefaction
of albumin outside the body, phenylaminopropionic acid breaks down into
phenylpropionic acid and phenylacetic acid. The former is not found as
such, in urine, but is combined with glycocoll as phenaceturic acid. In
this form it has been isolated from normal horse urine. Whether it occurs
in human urine, or not, has never been decided. If it were present to a
considerable extent, one might, aside from the possibility of its formation
from other sources, e.g. from the decomposition products of tyrosine, and
its formation during the decomposition processes in the tissues, draw the
conclusion that albumin, or a higher complex of amino acids such as a
polypeptide, is attacked by the bacteria of putrefaction. The increased
appearance of the other two acids would be an indication of intense putre-
factive changes taking place in the intestines.

Tyrosine and phenylalanine have also been supposed to participate in
the production of hippuric acid. If so, they would furnish the benzoic
acid radical. According to the above, it is evident that phenylalanine can
hardly play a part here, — at least, as far as it is a question of putrefactive
processes in the intestines. Tyrosine alone is to be considered in this con-
nection. It is, of course, possible that phenylalanine, and tyrosine as well,
are used for the production of hippuric acid in the metabolism of the cell.

ALBUMINS OR PROTEINS. 257

In this connection we would add, tliat two other acids, closely related
to tyrosine and phenylalanine, namely, homogentisic acid and uroleucic
acid, have also been found in rare cases in urine. The former is a dihy-
droxyphenylacetic acid:

CfiHs-OH

^CHa.COOH

the latter, a dihydroxyphenyllactic acid. They are both found during an
abnormal metabolism in the so-called " alcaptonuria."

Let us turn from our discussion of the tyrosine and phenylalanine de-
composition products to the sulphuric acid esters. We have already con-
sidered the occurrence of indoxyl- and skatoxyl-sulphuric acids in urine.
Intestinal putrefaction of albumin produces indole:

CH

HC C— CH

I II II

HC C CH

•^ / \ /
CH NH
and skatole (methvl-indole) :

CH
// \
HC C— C . CH3

I II H»

HC C CH^

■5*. / \ /
CH NH

They are oxidized in the tissues to indoxyl:

CH

// N

HC C— C(OH)

I II II

HC C CH

■^ / \ /
CH NH

and skatoxyl:

CH

HC C— C . CH3

I II II

HC C C(OH)

•^ / \ /
CH NH

and then combined with sulphuric or glucuronic acids.

We will at once state that skatoxylsulphuric acid has not been detected

258 LECTURE XII.

with sufficient certainty in urine. It is even problematical whether
skato.xyl occurs in urine. The suggestion has been made that it is present
in combination with glucuronic acid. At any rate, its presence is only
indirectly established. Indoxylsulphuric acid is found as an alkaline salt
in urine. It is the source of most of the urine indigo. Jaffe ' was the
first to discover that indoxylsulphuric acid resulted from the combination
of indole and indoxyl with sulphuric acid. He injected indole subcuta-
neously into dogs, and then found large amounts of indoxylsulphuric acid
in the urine. The suggestion has also been advanced that the indoxyl-
sulphuric acid in the urine is not only produced by intestinal putrefaction,
but that indole and indoxyl are also formed in the tissues. The researches
of A. Ellinger and M. Gentzen ^ have made this improbable. These
authors showed first of all that tryptophane, which is skatoleaminoacetic
acid, is the antecedent of indole, and the latter is formed from it during
putrefaction. When tryptophane was introduced directly into the small
intestine of rabbits, large amounts of indoxyl quickly appeared in the
urine. When, hovyever, the tryptophane was injected subcutaneously,
no indoxyl could be detected in the urine. That the excretion of indoxyl-
sulphuric acid in the urine is very intimately connected with the intestinal
putrefactive changes, is shown by the fact that when there is an intestinal
stoppage, especially in the small intestine, the quantity quickly increases.
Observations with fasting carnivorous animals have shown that the elimi-
nation of indoxyl continues, and the inference was drawn that indole and
indoxyl were also produced by the tissues. F. Miiller^ has, however,
shown that, during fasting, only the intestinal contents gave an in-
tense indole test, whereas the organs showed this only to a slight extent.
He, therefore, concludes that indole in the urine is exclusively of intestinal
origin. A limitation is certainly necessary, and this applies also to the
phenols. Putrefactive products can, of course, also be formed in other
parts of the organism, aside from the intestines, wherever puti-efactive
processes are at work; for instance, in putrid empyema, decomposing
tumors, etc.

If we add to urine an equal amount of hydrochloric acid containing a
little free chlorine or ferric chloride, a blue coloring matter is produced,
which can be shaken out with chloroform. This is indigo-blue:

C6ri4 C = C C6H4

^NH^ ^NH^

' Jaff4: Z. med. Wiss. 1872 and 1875.

' A. Ellinger and M. Gentzen: Hofmeister's Beit. 4, 171 (1903).
' F. Miiller: Mit. Wurzburger med. Klinik, 2, 1SS6; A. Ellingsr: Z. physiol. Chem.
39, 44 (1903).

ALBUMINS OR PROTEINS. 259

It is produced by the decomposition of indoxylsulphuric and indoxyl-
glucuronic acids into their components, and the simultaneous oxidation of
the indoxyl to indigo-blue. This coloring matter occurs in the indigo
plant in the form of a glucoside, called indican.' Indigo-blue can occa-
sionally be observed on the surface of putrid urine as a copper-red scum,
with a metallic luster. There is also another coloring material present,
an isomer of indigo-blue. This is indirubin, indigo-red. This coloring
matter has also been assumed to be related to skatoxyl, although this
substance has not yet been isolated, as such from normal urine. This
question must be left open for the present.

In the putrefaction of tryptophane, skatoleacetic acid and skatolecar-
boxylic acid are also produced. Up to the present time, only the latter
has been shown to be probably present in urine."

Furthermore, cadaverine and putrescine are also to be mentioned among
the putrefactive products of the intestines. Cadaverine is formed with
loss of carbon dioxide from lysine (diaminocaproic acid) , and is a penta-
methylenediamine.
CH, . CH2 . CH2 . CH2 . CH . C00H = CH2 . CH2 . CH2 . CH3 . CH2 + CO2

r II I

NH, NH2 NH2 NH2

Lysine Cadaverine

Putrescine, tetramethylenediamine, is produced from ornithine, diamino-
valeric acid, a constituent of arginine:

CH2 . CHo . CH, . CH . COOH = CH, . CH, . CH2 . CH2 + CO2

I " " I I I

NH, NHo NH2 NH2

Ornithine Putrescine

Phenylethylamine is produced from phenylalanine:

CeHs . CH2 . CH (NH2) . COOH = C6H5 . CH2 . CH2 . NH2 + CO2
and hydroxyphenylethylamine from tyrosine:
^OH ^OH

C6H4 = C6H4 + CO2

^CH2.CH(NH2)COOH ^CHs-CHa-NHa

Recently ^ the attempt has been made to trace the production of phenyl-
ethylamine and of hydroxyphenylethylamine to the action of trypsin or
of pepsin. A great many experiments have been carried out, with pure
pancreatic and gastric juices, taking great care to prevent any bacterial

' The indoxyl of urine is also wrongly called indican.
2 E. Salkowski: Z. physiol. Chem. 9, 1, 23 (1885).
' R. C. Emmerson; Hofmeister's Beit. 1, 501 (1902).

260 LECTURE XII.

contamination, without finding any ground for such an assumption. That,
on the contrary, carbon dioxide is split off by the action of putrefactive
bacteria, has been convincingly shown by Ellinger.' Cadaverine and
putrescine are only found in the faeces and urine under exceptional condi-
tions, as, for example, in dysentery and acute enteritis. It is particularly
well known that these diamines appear in cystinuria, a disturbance in the
metabolism of albumin which we shall soon take up more in detail. It is
still questionable what the relation is between the appearance of these
two diamines and this metabolic irregularity. At all events, these com-
pounds are not always observed when cystine is eliminated in the urine.
We have now mentioned all those products of urine which are to be
traced to putrefaction in the intestines, and will now turn our attention
to an acid which has been found only in the urine of certain animals,
especially dogs, namely kynurenic acid, whose mother-substance has been
quite recently recognized to be tryptophane. It is y -hydro xyquinolin-/?-
carboxylic acid: ^

CH C (OH)

HC C C . COOH

HC C CH

^CH^ ^N^

The formation of kynurenic acid from tryptophane was proved by
Ellinger, who fed some of the tryptophane, inclosed in a gelatine capsule,
to a dog, and estimated the amounts of kynurenic acid before and after
the feeding. The increase caused by tryptophane, was very appreciable.
Rabbits, which ordinarily do not excrete any kynurenic acid, did so after
the administration of tryptophane.^ Human beings, on the other hand,
were not found to produce any kynurenic acid.

These decomposition products give us a very good idea of the protein
decomposition in the tissues. We will not go very far astray if we accept
the following conception of the behavior of the proteins in the animal or-
ganism. In the stomach, the albuminous substances are almost entirely
broken down into a number of very complicated products by the action of
pepsin and hydrochloric acid. These pass on to the intestine, where they
are attacked further by trypsin. Under these influences, polypeptides are
produced, a larger or smaller number of the amino acids entering into
their composition. A large number of free amino acids are split off at
the same time, first, tyrosine, tryptophane, and cystine. Then follow

A. Ellinger: Z. phy.siol.Chem. 29, 34 (1900); Ber. 31, 318.3 (1899); 32, .3542 (1900).
J. Liebig: Ann. 86, 125 (1853). R. E. Swain: Am. J. Physiol. 13, 30 (1905).
A. Ellinger: Z. physiol. Chem. 43, 325 (1904).

ALBUMINS OR PROTEINS. 261

alanine, leucine, glutamic acid, aspartic acid, lysine, arginine, histidine,
etc. Phenylalanine and proline are undoubtedly present in combination,
for they are unacted upon by trypsin. They remain unattacked, com-
bined with other amino acids in the form of polypeptides. All these
cleavage-products are absorbed. The albumin synthesis starts in the
intestinal walls, the serum-albumins being first formed. The rapidity of
this synthesis is dependent on the presence of certain amino acids, for, as
recent investigations have proved, the relative amounts of the amino acids
in the serum-albumins are very constant. The albumin synthesis would
necessarily be regulated by the amount of that amino acid which is present
to the smallest relative extent. There is always the possibility that one
amino acid may change into others. This is known to be true only of
amino acids of the aliphatic series, among themselves; but in the same
manner we can imagine the production of aromatic amino acids from one
another. On the other hand, it is hardly probable that relations exist
between these two groups or at most only in the sease that an aromatic
amino acid might give rise to an aliphatic one, or at least, a fatty acid.
Our knowledge of the formation of one food-stuff from another,' e.g. the
production of fat from the carbohydrates, forces us to admit the pos-
sibility of several amino acids being produced from a single one.

If such transformations take place then naturally the extent of the
albumin synthesis is considerably widened. Those amino acids which are
not utilized for the production of new proteins are evidently already
broken down in the walls of the intestine; i.e. the amino group is first
split off and the residue consumed. The ammonia thus formed prob-
ably is utilized in the production of urea. From these conceptions, we
should expect to find a 'priori, that the various albuminous substances
behave differently; i.e. under certain conditions an influence upon the
extent of the synthesis of albuminous substances in the intestines might
affect the entire metabolism. This is well shown in the case of gelatin,
which not only lacks whole groups of amino acids, buf at the same time
possesses combinations of such, which affect unfavorably the breaking
down of the molecule and indirectly the formation of new proteins. The
other proteins might also, according to the amino acids in their composition,
be more or less favorable to the synthesis of protein, and especially for
the formation of the serum-albumins. It could even be expected that it
would not be possible to maintain the same nitrogen balance with every
albuminous substance. Here it is assumed that the amino acids which
are already broken down in the intestines are to a certain extent eliminated
from the albumin metabolism.^ We have intentionally dwelt on these

CT. Lecture XIV.

Cf. Lecture XI, p. 225.

262 LECTURE XII.

relations somewhat more in detail, because they have hitherto received
but little attention in experimental investigation.

The albuminous substances now produced, together with the ammonia
that has been split off, and, possibly, other cleavage-products from the
amino acids, pass from th'e intestine to the liver, and from here into the
general .circulation. We will mention that the liver is quite generally
considered as the place where the above-mentioned aromatic products of
putrefaction are conjugated with sulphuric and glucuronic acids. We
have already mentioned the fact that urea is formed in the liver on a large
scale.

As a consequence of the extensive decomposition and reconstruction of
proteins in the alimentary tract, only those albuminous substances circu-
late in the organism which correspond to its entire construction. Every
cell continually receives the same nourishment in the same composition,
through the instrumentality of the blood. The whole mechanism of
the cell is thereby greatly simplified. The cell is, in the widest sense,
independent of the nature of the food in the exercise of its functions.
It is, in its entire metabolism, adjusted to a nourishment of quite definite
composition, which is always available to satisfy its demands. A prom-
inent part is taken by the intestine in the total metabolism. The
nutritional relations of the entire cell-material depend in the widest sense
upon its activity. Its functions are simplified, in proportion as the
proteins from the food are prepared by the combined action of hydro-
chloric acid and pepsin and of trypsin. The cells of the intestines will
be built up more quickly the better the material available for synthesis
is suited for the new proteins. Although any derangement in the
secretion of the ferments would undoubtedly affect the processes in the
intestinal tract, the disturbance in this tract must affect the entire meta-
bolism even more seriously.

The individual body-cell removes from the blood such protein sub-
stances as it requires. It breaks them down in the same manner that
trypsin does. Amino acids result, which, on further decomposition,
produce, on the one hand, urea, and, on the other, carbon chains free
from nitrogen, of whose natm-e we still know but little, and which,
perhaps, enter into relations with the carbohydrates, fats, and, possibly,
other organic components of the tissues. The breaking down of the
amino acids in the cells is still an obscure process. We only know that
the total nitrogen soon appears in the urine. It is questionable whether
the total combustion of the amino acids is immediately effected after the
elimination of the amino or CO . NH2 groups, or whether the surviving
carbon chains in their further decomposition are independent of the above
process. The formation of the nitrogenous end-products, whether it be
urea or uric acid, is also but imperfectly explained. If we assume that the

ALBUMINS OR PROTEINS. 263

liver is practically the only organ in the body producing urea, we must
conclude that the nitrogenous cleavage-products, whether ammonia or
any compound containing the CO . NHq group, formed by the cell func-
tions, would have to be transported to the liver, and there acted upon.
Here is another large gap in our knowledge concerning the decomposition
of albumin in the tissues, which we do not seem able to bridge over at
present. Hypotheses have, therefore, been advanced here, which we
have already discussed under the formation of urea and uric acid.

If we accept the foregoing e.xplanation of the decomposition of albumin
in the tissues, we must naturally expect that the presence of some of the
amino acids which have been designated as representing transition stages
may be detected. As a matter of fact, certain observations do indicate
the presence of amino acids in the intermediate metabolism of albumin.
We will, moreover, state, in order to prevent any misapprehension, that
when the proteins are broken down by the cells into the amino acids,
further decomposition need not necessarily immediately follow, any more
than that the muscles must immediately consume any dextrose which
may be presented to them. Just as the muscles produce their glycogen
from dextrose, so the cells undoubtedly utilize the decomposition-products
according to their requirements, at one time decomposing them further,
and at another time linking them together into chains, thus utilizing the
proteins thereby formed as building material for the contents of their
own cells, or for forming new cells. Every individual cell must build up
its own albumin, in the same manner as the intestine, and form its
own peculiar albumin from its own particular nourishment, the serum-
albumin. This probably takes place in much the same way as in the
intestine, with its digestive ferments furnished by the glands. The
body cells, also, probably supply themselves with the necessary materials
for their cell requirements by breaking down the proteins into simpler
portions. Polypeptides and amino acids very likely appear as transition
products in the intermediate metabolism of albumin. Just as the intes-
tines do not decompose the albumins entirely into the lowest cleavage-
products, so we need not expect the tissue-cells to decompose all of the
albumin into its simplest components. These cells also pi-obably only
decompose the proteins to the point where they can be utilized to recon-
struct new albumins.

The assumption that cell-metabolism also produces amino acids, has
been supported by the fact that it is possible to protect certain of these
amino acids from being acted upon further by the administration of
specific compounds which possess the faculty of uniting with them, and
thus to recover them. In this way the presence of cystine in dogs has been
verified by introducing phenyl halides (brom-, chlor-, or iodo-benzene)
into the animal. By feeding benzoic acid to mammals, we obtain a com-

i

264 LECTURE XII.

pound of glycocoll, while from birds we get one of ornithine. Some of the
amino acids, such as cystine, have been directly isolated from normal
organs.

The question whether amino acids are normal constituents of urine, has
recently been raised repeatedly. Various answers have been given. If we
examine critically the investigations so far published, we shall have to
admit that there is no positive proof yet brought forth indicating the
presence of amino acids in urine under normal conditions. Glycocoll is
the only one that has been identified positively, and this was only accom-
plished after the urine had been liberally treated with alkali for many
hours; in fact, several days. We can easily imagine that the glycocoll
may have been split off from some compound. Until it is possible to
show that the amount of this product depends upon the extent of albumin
decomposition in the organism, we cannot regard this discovery as proof
of the appearance of glycocoll in cell-metabolism. It is noteworthy that
only glycocoll has so far been isolated. This amino acid is utilized to a
considerable extent for conjugation with aromatic substances, especially
benzoic acid. We can easily imagine that the glycocoll found in urine
originated from this source. It is very probable that the organism main-
tains a supply of glycocoll for just this coupling proce.ss. When we con-
sider in addition that the kidneys are active producers of hippuric acid,
we can appreciate the possibility of glycocoll being flushed into the urine
under certain conditions. From the investigations at hand, we are not
at all justified in stating that amino acids are normal constituents of
urine.'

Amino acids are, however, often present in urine in large amounts under
certain pathological conditions. This, for instance, occurs in the case of
acute atrophy of the liver, a disease in which the albumin decomposition
is very rapid. The liver, in this case, is flabby and emaciated. The con-
tents of the capsule of Gli.sson are quite soft, and, in part, semi-fluid. An
extensive destruction process has taken place in all the liver cells. Amino
acids can be found in the liver-paste, leucine and tyrosine being easily
isolated on account of their insolubility. The remaining elements of the
albumin molecule, especially those easily split off, are also probably present.
The two amino acids mentioned, often crystallize out directly on the liver
itself, in the form of a white coating. Leucine and tyrosine have been
found in the urine at such times. A very analogous condition arises in
phosphorus poisoning. Here, again, we find amino acids in the urine;
tyrosine, leucine, and glycocoll ' having been isolated. Undoubtedly

' E. Abderhalden and A. Sehittenhelni: Z. physiol. Chem. 47, 1906. G. Embden and
H. Reese: Hofmeister's Beitr, 7, 411 (1905).

' E. Abderhalden and P. Bergell; Z. physiol. Chem. 39, 464 (1903). E. Abderhalden
and L. F. Barker: ibid. 42, 524 (1904).

ALBUMINS OR PROTEINS. 265

there are other albuminous decomposition-products present in the urine
of animals which have been poisoned by phosphorus.

We will add that the sudden destruction of cell-albumin as it occurs
in acute atrophy of the liver, phosphorus poisoning, and many other con-
ditions, has been compared to the autolysis of dead tissues. By autolysis,
we mean " self-digestion " of the organs, which follows in a short time,
when these are preserved in a sterile condition. A gradual solution and
liquefaction of the whole organ takes place. Among the end-products of
this process we find, for one thing, decomposition products of albumin, —
arginine is rarely present, as it is further decomposed by the arginase, — ■
then again cleavage-products from the nucleins, and finally also "com-
pounds arising from the remaining elements of the tissue. We obtain
the impression, that all cell-ferments become active immediately after
death, and then, when all the regular functions have ceased, indiscrimi-
nately tear everything to pieces. It is correct to assume from this con-
ception of autolysis, that an analogous fermentation occurs in the cells.
It would be, however, a grave error to conclude that the autolytic
decomposition is to be regarded as the normal breaking down of the cell.
The living cell unquestionably does not permit all its ferments to act at
one time. It regulates its metabolism most carefully. One fermentation
process is carried out, another then follows. By one of these processes
a certain cleavage-product is formed, while the action of another ferment
breaks it down further. All these processes cooperate with one another.
The reconstruction proceeds uninterruptedly together with the decompo-
sition. In the dead tissues all this regulating mechanism is wanting.
Decomposition alone takes place. We are not justified in considering the
severe destruction which occurs in the above-mentioned liver tissues as
parallel to autolysis. It is possible that the whole process is a similar
one, that the solution of the cell structure precedes the tleath of the cells;
on the other hand, it must be remembered that we have, as yet, only
established a restricted decomposition of cell proteins, while an absolute
confirmation of the total dissolution of the cell tissues, which characterizes
autolysis, is missing. The cell destruction, under the pathological condi-
tions mentioned, is also much more rapid in autolysis than under normal
conditions.

Autolysis also seems to play a part in the living organism, — in fact,
assisting in the removal of dead matter; for example, of the fibrin produced
by pneumonia in the lungs;' in the reduction of the uterus after childbirth ;
very probably in the absorption of copious exudations of corpuscular
elements and the decomposition of decaying neoplasms, which have been
ctit off from the circulation, etc. It is questionable whether we are justi-

' F. Miiller: Verb. XX, Kon. Med. Wiesbaden, 1902.

266 LECTURE XII.

fied in calling these processes autolytic. We only know that the organism
is capable of mobilizing ferments which take care of foreign material, and
by decomposing and reducing the complex molecules, prepare it for assimi-
lation. In fact, in pneumonia, during r&solution the bronchial tubes seem
to possess functions very analogous to those of the intestine. It would
be better, for the present, to restrict the term " autolysis " to the ferment
action of the cells in the tissues, which follows some time after death.
It is like the works of a clock, whose spring has been released and sud-
denly runs down.

Amino acids have recently been found in the urine during various dis-
eases. If we summarize these observations, we will obtain the impression
that the metabolism has been deranged by lack of oxygen. Thus, tyrosine
is found in the urine after prolonged, deep narcosis, during the coma of
a diabetic, etc'

While these cases represent merely isolated cases of the appearance of
individual amino acids, due to temporary derangements, and which are not
at all permanent, we, however, also know of a derangement in metabolism
in which a greater or less amount of an amino acid is always present in
the urine. This occurs during cystinuria. Cystine- is found in the urine
during this rather rare disturbance in the decomposition of albumin.
Small amounts of this compound seem to be always present in urine.' In
cystinuria, however, the quantity is very largely increased, and often
leads to the formation of calculi. There is not the least doubt but that
this cystine originates from albumin. It, like the albuminous cystine, is
an a-diamino-/3-dithiodilactylic acid. Emil Fischer and Umetaro Suzuki *
have recently established the identity of the two substances.

The significance of cystinuria was long in doubt. The discovery of
L. von Udransky and E. Baumann ^ that other di-amines (putrescine and
cadaverine) are present in the urine during cystinuria, for a long time led
to the assumption that cystinuria is caused by an increased intestinal putre-
faction. Cystine, according to this assumption, is split off from albumin
in the intestines and absorbed as such. To-day we know that the forma-
tion of amino acids is a normal function of the alimentary tract, in no case
causing their elimination in the urine. The di-amines mentioned are by

' E. Abderhalden: Z. physiol. Chem. 44, 17 and 40 (1905); 45, 468 and 471 (1905).

' W. F. Lobisch: Ann. 182, 231 (1876). A. Niemann; Peut. Arch. klin. Med. 18,
232 (1876). W. Ebstein: 19, 138 (1877); 30, 594 (1882). B. Mester: Z. physiol. Chem.
14, 109 (1890). A. Loewy and C. Neuberg: ibid. 43, 338 (1904). C. Alsberg and O.
Folin: Am. J. Physiol. 14, 54 (1905). E. Abderhalden: Z. physiol. Chem. 38, 557 (1903).
E. Abderhalden and A. Schittenhelm: ibid. 45, 468 (1905).

' Stadthagen: ibid. 9, 29 (1885). E. Goldmann and E. Baumann: ibid. 12, 254
(1888).

* E. Fischer and V. Suzuki: ibid. 45, 405 (1905).

» L. V. Udransky and E. Baumann: Z. physiol. Chem. 13, 562 (1889).

ALBUMINS OR PROTEINS. 267

no means found in all cases of cystinuria. It is far more probable that the
disease is to be regarded as a disturbance in the breaking down of albumin,
on the part of the tissues. That the cystine from the albuminous sub-
stances in the food is absorbed and assimilated is evident from the fact
that the albuminous material in the tissues of a patient afflicted with
cystinuria, certainly contains cystine; and that no diminiition in the
amount of this amino acid can be detected. Cystine is evidently produced
in the decomposition of proteins during cell-metabolism, and is not
further worked over. It is difficult to say why, in these cases, cystine
is not decomposed. A patient afflicted with cystinuria consumes any
cystine administered to him, and does not eliminate all the cystine-sulphur
as such. It would be easy to imagine some change in the cystine molecule,
such that the cell ferments are unable to find any point of attack. We
have seen that the cystine from albumin, and that of the urine, are
identical. This question must be left unsettled for the present. It may,
of course, be possible, that the ferments capable of decomposing cystine
are absent from some cells, and that this substance is, therefore, eliminated
unchanged. Such an assumption has not, however, been experimentally
confirmed. It would start with the hypothesis that each cell possessed
a distinct ferment to produce each different amino acid, or group of amino
acids. We must say that we have absolutely no proof of such a condition
of affairs. We can imagine that cystine might occupy an isolated position
on account of its difficult solubility. It is, however, possible that con-
ditions may exist in the cells of a person afflicted with cystinuria, which
may cause cystine to be thrown out. That cystine may, in time, accu-
mulate in the tissues to large proportions, has recently been proved in the
case of a boy 2U months old.^ He died with indications of gradual
inanition. A post-mortem examination showed all the organs permeated
with crystals of cystine. The spleen, for example, was saturated with
cystine, and from this organ the pure amino acid could easily be isolated
in large quantities. It was interesting that this was a case of inherited
cystine diathesis, — in fact, in a progressive form. Perhaps some light
may be shed upon this rare derangement in metabolism by the obser-
vation that other amino acids, besides cystine, may be found in the urine
during this disease.- Thus, in one case, cystine, leucine, and tyrosine were
found. Apparently from this discovery cystinuria corresponds to a more
general disturbance in the breaking down of albumins than is usually
assumed, and that, to a certain extent, this disease is to be considered as
the simplest form of such derangement. We must again state, however,

■ E. Abderhalden: ibid. 38, 557 (190.3).

' E. Fischer and U. Suzuki: Z. physiol. Chem. 46, 405 (1905). E. Abderhalden and
A. Schittenhelm: ibxri. 45, 468 (1905).

268 LECTURE XII.

that it is impossible to give a perfectly clear picture of the metabolic
disturbance in question, which shall be based upon our present experi-
mental knowledge. If we base our judgment concerning this anomaly in
albumin-metabolism not only upon the observations made upon those
afflicted with cystinuria, but upon our general knowledge of the total
metabolism of albumin in the tissues, it then appears as most probable
that we have in cystinuria a disturbance in the decomposition of proteins
in the cell-metabolism. Conversely, we can consider the appearance of
cystine in this disease as further evidence of the formation of amino acids
from albumin in the intermediate metabolism, always remembering that
one supposition is dependent on the other, and thus it is not a definitive
proof.

Our insight into the intermediate decomposition of albuminous bodies
is by no means limited to the discovery of amino acids in the urine
under specific conditions, and to the recognition of the final albumin
cleavage-products, — urea, in the case of mammals, and uric acid in birds
and fishes. There are other products present in the urine, as yet largely
unknown, which contain nitrogen and sulphur, and are undoubtedly
closely related to albumin-metabolism. We will disregard the fact that
there are albuminous substances in urine which have been variously inter-
preted. They are probably not simple substances. They belong partly
to the mucins, and in part to the group of nucleo-albumins, and probably
originate in the urinary passages. They have no bearing on the subject
of albumin-metal)olism. This applies especially to the large quantities
of albumin which appear in the urine under pathological conditions, and
especially in diseases of the kidneys. These only affect the albumin-
metabolism indirectly, inasmuch as they continually withdraw this valuable
material from the body, thus depriving the organism of the energy con-
tained therein. It is indeed possible, that an exact examination of these
substances would give us an insight into the course of albumin decompo-
sition in the tissues. It would certainly be of the greatest interest to
know the origin of the albumin always present in the various forms of
nephritis.' We usually assume that the serum-proteins (serum-globulin
and serum-albumin) under pathological conditions pass into the urine.
Although this assumption is very plausible, it must be said it does not
necessarily explain all the cases arising. That albumin does not normally
appear in urine, excepting, of course, in traces, is due to the fact that the
epithelial cells of the kidney, or those of the glomeruli, will not permit the
colloidal albumin to pass through. This simple explanation is not inva-
riably true, as is shown by the appearance of a very well-defined albumi-
nous substance, the so-called " Bence-Jones albumin," in the urine. It is

' E. .'Vbderhalden: Z. exper. Path. u. Therapie, 2, 642 (1905).

ALBUMINS OR PROTEINS. 269

principally present in cases of sarcoma formations in the bone marrow
{Sarcotnatosis ossium). It is usually found only in the urine, the epi-
thelia of the glomeruli permitting this substance to pass through, at the
same time retaining all the serum-albumins. It might be thought that
the Bence-Jones albumin represents a lower albumin, and consequently
diffuses through the urinary passages.' This assumption is, however, in-
correct, because this albumin contains all of the usual amino acids, which
seems to indicate that the albumin has not in any way undergone much
decomposition. Tyrosine is one of these amino acids, and this amino
acid, as is well known, is very easily split off from the rest of the molecule.
The objection might be raised, of course, that the tyrosine is linked in a
different manner in the Bence-Jones albumin from that in other varieties
of albumin. There is at present no ground for such an assumption.
According to its content of amino acids, the Bence-Jones albumin does
not correspond to either of the two serum-proteins, and may be con-
sidered as one of the tissue-albumins, which, without being broken down
or changed into one of the serum-albumins, is transmitted to the blood,
and then is probably eliminated as an albumin foreign to the blood
although suitable for the body. It would be interesting to investigate
other analogous albuminous excretions from the kidneys.

Although such products are only found under specific conditions, normal
urine, nevertheless, contains other compli<'ated compounds, whose nature
has not yet been determined, but which, from their elementary composition,
must be closely related to albumin-metabolism. Their high percentage
of oxygen stamps them as albumin oxidation products. Their presence
indicates the possibility that the disintegration of the albumin molecule
may proceed in different ways, and that our assumption, that albumin is
broken down in cell-metabolism through the amino acid stage, does not
apply to all albumin decomposition. It is indeed possible that a part of
the albumin is oxidized in a manner unknown to us without undergoing
previous cleavage. It is not impossible that, for this kind of albumin
decomposition, those complexes come into consideration which, as we
have seen, strongly resist the action of the proteolytic ferments. At all
events, we shall expect that when these products are explained, we shall
receive further insight into the intermediate metabolism. Here we can
only mention the names of these various compounds, and assert that no
proof exists of their individuality. Bondrizyski and Gottheb ^ distinguish

' E. Abderhalden and O. Rostoski: Z. physiol. Chem. 46, 125 (1905). Cf. A.
EUinger: Deut. Arch, kliii. Med. 62, 255 (1899). A. Magnus-Levy: Z. physiol. Chem.
30, 200 (1900). F. Reach: Deut. Arch. klin. Med. 82, 390 (1905).

' St. Bondzynski and Gottlieb: Zentr. Med. Wiss. (1897) No. 33, 577. St. Bondzyiiski
and Panek: Bull, de r.\cad. d. sciences de Crascovie, Oct. 1902. St. Bondzyiiski, St.
Dombrowski.and K. Panek: Z. physiol. Chem. 45,83 (1905). F. Pregl: Pfluger's Arch.
75, 87 (1899).

270 LECTURE XII.

first of all between an oxy -proteinic acid and an alloxy-proteinic acid.
Antoxy -proteinic acid has recently been added to these. All three acids
contain sulphur, nitrogen, and large amounts of oxygen.

The following figures will give some idea of their composition. The
antoxy-proteinic acid contains 43.21 per cent C, 4.91 per cent H, 24.40 per
cent N, 0.61 per cent S, and 26.33 per cent 0; the oxy-proteinic acid .39.62
per cent C, 5 . 64 per cent H, 18 . 08 per cent N, 1.12 per cent S, and 35 . 54
per cent 0; and the alloxy-proteinic acid 41 .33 per cent C, 5.70 per cent
H, 13.55 per cent N, 2.19 per cent S, and 37.23 per cent 0. We must
also add that O. Thiele ' has described a uroferric acid occurring in urine,
which undoubtedly belongs to this group of compounds. By heating it
with hydrochloric acid in a sealed tube it decomposes, producing melanin
substances, carbon dioxide, ammonia, organic sulphur compounds, hydro-
gen sulphide, and aspartic acid. We must also add that these substances
do not give any albumin reactions. The biuret test, Millon's reaction,
and the remaining characteristic test for albumins and their closely-
related cleavage-products, all give negative results. We must content our-
selves for the present with the mere enumeration of these substances. It
is possible that perhaps some light is thrown upon the formation of them
by the fact that a difficultly-dialyzable body containing no amino acids ^
may be isolated from urine, in the same way as alloxy-proteinic acid
and oxy-proteinic acid were obtained. Such acids may, however, be
obtained from this substance by boiling it with concentrated hydrochloric
acid. Glycocoll, leucine, and glutamic acid are then isolated, and the
presence of phenyl-alanine and aspartic acid indicated. The substance
did not contain any tyrosine. It is very probable that this product is
a residue of a partially disintegrated albumin molecule, which has escaped
further disintegration. We know nothing further about its relations with
the other acids just mentioned.

In the discussion of the decomposition products of tyrosine and phenyl-
alanine, we called attention to two hydroxy-acids which occur in the urine
during the very rare metabolic disturbance known as alcaptonuria. Alcap-
ton was the name which Bodeker ' gave to a substance which he isolated
from the urine of a man afflicted with diabetes. It gave to the urine two
distinguishing characteristics. The urine showed a very appreciable reduc-
ing power, and had the property of turning dark brown or black, taking on
oxygen, when alkali was added. This aleapton has been isolated from
urine by M. Wolkow and E. Baumann,* and its composition ascertained

■ O. Thiele: Z. physiol. Chem. 37, 251 (1903).
' E. Abderhalden and F. Pregl: ibid. 46, 19 (1905).
' Bodeker: Z. rat. Med. 7, 130 (1859); Ann. 17, 98 (1861).

* M. Wolkow and E. Baumann: Z. physiol. Chem. 15, 228 (1891). Here are given
references to the older literature.

ALBUMINS OR PROTEINS. 271

after Kirk ' had previpusl^y shown that a crystalHzed acid could be obtained
from the urine of three children in the same family. He soon recognized
the fact that it was a mixture of uroleucic acid and uroxanthic acid. The
latter is, undoubtedly, identical with the homogentisic acid of Wolkow and
Baumann, who have established its constitution.^ It is a di-hydroxy-
phenyl-acetic acid. Uroleucic acid, on the other hand, is a di-hydroxy-
phenyl-lactic acid. The latter has only occasionally been found in alcap-
tonurie urine, and is, undoubtedly, the antecedent of homogentisic acid.
Wolkow and Baumann have discovered the source of these acids, and also
the conception of the whole phenomenon. Alcaptonuria is not to be looked
upon as a disease; it is more to be considered as indicative of an anomalous
metabolism, which, without causing any noticeable derangement, may
continue for the entire lifetime. It is of considerable interest to note its
appearance in several members of the same family. As far as the origin
of the homogentisic and uroleucic acids are concerned, it is natural to
look to the aromatic groups derived from the albumin molecule. We
have already called attention to the fact that a large number of decompo-
sition products may be obtained directly from this source and appear in
the urine.

Tyrosine, until recently, was the onlj' elementary, aromatic constituent of
albumin, which was invariably found present and easily obtained. From it
are derived 7>hydroxy-phenyl-propionic acid, p-hydroxyphenylacetic acid,
p-cresol and phenol. Wolkow and Baumann, by means of feeding experi-
ments, showed that the acids of alcaptonuric urine were also formed from
tyrosine. They found that the administration of tyrosine to a man afflicted
with alcaptonuria caused an appreciable increase in the amounts of alcap-
ton acids excreted. Wolkow and Baumann also indicate phenyl-alanine
(phenjd-amino-propionic acid )' as another source of these alcapton acids.
These investigators did not have a sufficient quantity of this aromatic acid,
and they had to confine their researches to the relation of the alcapton
acids to tyrosine. The methods recently introduced by Emil Fischer,
for the isolation of the cleavage-products of proteins, made it possible not
only to obtain phenyl-alanine in larger quantities, but also showed its
universal distribution as an elementary constituent of .albuminous sub-
stances. Very little albuminous material is free from it, and it is even
more widely distributed than tyrosine. On the basis of this knowledge,
and because of the recent discovery of the easy accessibility of phenyl-
alanine, the relations between it and the alcapton acids have been inves-
tigated anew. W. Falta and Leo Langstein * found that when this amino

Kirk: Brit. Med. Jour, 2, 1017 (1886); J. Anat. and Physiol. 23, 69 (1889).

E. Baumann and S. Fraenkel: Z. physiol. Chem. 20, 219 (1894).

M. Wolkow and E. Baumann: loc. cit. p. 266.

W. Falta and L. Langstein: Z. physiol. Chem. 37, 513 (190.3).

272 LECTURE XII.

acid was administered to a man afflicted with alcaptonuria, the elimination
of the homogentisic acid increased in the same manner as when tyrosine
was used. Both of these elementary aromatic constituents of albumin
are, therefore, to be considered as forming the basis for the formation of
the alcapton acids.' It is very important to note that as far as our present
knowledge is concerned, all the phenyl-alanine and tyrosine in the food
materials are converted by persons afflicted with alcaptonuria, into the
alcapton acids, so that the disturbance in the disintegration of these amino
acids seems to be very complete.

A comparison of the constitution of tyrosine and of phenyl-alanine with
that of the alcapton acids shows us that the formation of the latter from
the former is not an altogether simple process.

V
CH2 . CH . NH2 . COOH CH2 . CH . NH2 .COOH

phenyl-alanine Tyrosine

VOH VOH

CH2 . CH(OH) . COOH CH2 . COOH

Uroleucic acid Homogentisic acid

Tyrosine is para-hydroxyphenyl-a-aminopropionic acid, and phenyl-
alanine is a phenyl-a-aminopropionic acid. All the decomposition products
of tyrosine which we have met with, partly as putrefactive products, and
partly as products arising from intermediate metabolism, belong, as
tyrosine itself does, to the para compounds. The constitutional formulae
of homogentisic and uroleucic acids, as shown above, indicate that this
is not true of them. It is difficult to understand the formation of both
of these alcapton acids from any of the known aromatic components of the
proteins. In the transformation of tyrosine into homogentisic acid, the
hydroxyl group must certainly be eliminated, either by being split off
or by migrating. ' Two other places in the benzene ring are then oxidized,
hydroxyl groups being formed para to one another. Altering the side-
chain of the amino-propionic acid into an acetic acid residue presents
nothing unusual, and can easily arise by merely removing the amino group.

It is veiy probable that the homogentisic acid is not directly produced
from tyrosin, but from its derivative, /j-hydroxyphenylacetic acid. It is
here evidently that the anomaly in the further degradation of tyrosine
occurs. The corresponding compound from phenyl-alanine is phenylacetic

Cf. also A. C. Garrod and T. S. Hele: J. Physiol. 33, 198 (1905).

ALBUMINS OR PROTEINS. 273

acid. Embden ' has shown that the alcapton acids are not produced when
the latter is administered. This investigation does not, however, pre-
clude the possibility that phenyl-acetic acid is one of the first degradation
products of phenyl-alanine, even from persons affected with alcaptonuria,
or that the disintegration of this amino acid does not proceed abnormally
from the very beginning. Efforts have been made to estalalish homogen-
tisic acid as a normal intermediate cleavage-product of phenyl-alanine
and tyrosine." From this point of view, alcaptonuria would be looked
upon as a check on the complete combustion of the benzene nucleus. The
formation of homogentisic acid would then be looked upon as an oxidation
preceding the disruption of the benzene ring. The person afflicted with
alcaptonuria would not be capable of carrying this process to the end, as
a result of which, this anomalous metabolism produces a decomposition
product in the intermediate metamorphosis which would otherwise have
been lost to us.

There is something very attractive in the suggestion that alcaptonuria
acts as a simple restraining influence in the normal disintegration of tyrosine
and phenyl-alanine. We must admit that this assumption has received
some support in the researches of Otto Neubauer and W. Falta. On the
other hand, we must remember that the manner of formation of the
alcaptonuric acids still remains a hypo.thesis, and that no absolute proof
of its truth has as yet been presented.

The place of formation of the alcapton acids in the organism of a
patient afflicted with alcaptonuria was for a long time very much in
question. Wolkow and Baumann claimed that they were produced in
the upper part of the intestine, by the aid of micro-organisms. We
to-day believe that the alcapton acids are probably formed in the
tissues themselves. This conclusion follows from the fact that phenyl-
alanine, as far as known, is not set free by proteolytic ferments in the
alimentary tract. The conditions are different with tyrosine. Large
quantities of this are set free in the intestine. This, under normal con-
ditions, is very largely assimilated, being even utilized by those affected
with alcaptonuria in the production of albumin, which is indicated by
the fact that the albuminous components of their blood show the same
amounts of tyrosine and phenyl-alanine, as do those of normal persons.^
A small portion of the tyrosine is undoubtedly attacked by bacteria in the
intestine, in this way producing the various decomposition-products, until
phenol is reached. The intermediate products, 73-hydroxyphenylpropionic
acid, and p-hydroxyphenylacetic acid, may also act as sources for the pro-

' H. Embden: Z. physiol. Chem. 18, .304 and .317 (1894).

' L. Gamier and S. VoLsin: .Arch, physiol. Ges. 5, 224 (1892). 0. Neubauer and
W. Falta: Z. physiol. Chem. 42, 81 (1904).

' E. Abderhalden and W. Falta: Z. physiol. Chem. 39, 143 (1903).

274 LECTURE XII.

duction of the alcapton acids. The largest amounts are, however, produced
in cell-metabohsm, afte^r the cell has disintegrated the proteins into their
components, and now completes the decomposition of the amino acids.
This is where the anomaly, or restriction, occurs.

If we combine all our knowledge of the proteins, their composition, and
their decomposition, with what we know experimentally about the diges-
tion, absorption, and assimilation of the albuminous materials of our food,
together with the information gleaned from our study of their changes
and their final disintegration in the tissues, we shall find that we are
obtaining a very good idea of the whole subject of albumin-metabolism.
To be sure, many bridges have been built purely provisionally from analo-
gous conclusions and probable relations, to enable us to pass from one
well-founded principle to another, and hypotheses still permeate all of
our views. We, however, do not doubt that the progress of albumin
chemistry will strengthen one position after another, and that eventually
facts will supplant our assumptions.

LECTURE XIII.
THE NUCLEOPROTEIDS AND THEIR CLEAVAGE-PRODUCTS.

In discussing the proteins, we have only briefly referred to those which
do not occur by themselves, in the tissues, but are united to a second
atomic complex. To these compound proteins, also called proteids, belong
the nucleoproteids. They occupy an important position, not only in animal
economy, but in that of the plant cell as well. They are widely distrib-
uted, and are mainly found in the nuclei of cells. It is at present very
difficult to decide whether the materials classified under the name of
nucleoproteids are of an individual nature, within certain limits. They
are purified with difficulty, and are mainly characterized by their cleavage-
products. From what we know, it appears that the albuminous compo-
nent may vary widely in character. For example, we find histones and
varieties of protamines. The other component, which we shall shortlj'
consider, also shows differences in composition according to the nature
and derivation of the nucleoproteid. When we recollect all that has been
said regarding this class of substances, we are involuntarily forced to the
conclusion that an exact decision as to the construction of the nucleo-
proteids is not possible, largely because it is certain that these proteids are
obtained in various degrees of purity, according to the method used for iso-
lating them; or, perhaps better e.xpressed, because in certain investigations
products have been worked with which had already undergone considerable
change. The albumin component shows all the characteristics common to
the proteins. Above all, it possesses the property of "denaturizing," which
often serves to impart an entirely new property to an isolated product,
thus apparently indicating a new compound. We are forced to obtain
the nucleoproteids from the cells themselves, i.e., from a very complex
mixture of proteins. The fact that in the different nucleoproteids the
two components are combined with different degrees of firmness, may
likewise lead to errors, and prevents, more than anything else, any energetic
attack in the attempt to purify the isolated products.

When we take all these facts into consideration, there is little wonder
that the existence of the nucleoproteids should be repeatedly questioned.
We have to thank F. Miescher for much of our knowledge about these
bodies. The non-albuminous component, nucleic acid, will precipitate
albumin. It is conceivable that it shows its precipitating power during
the isolation of the proteids, and is thus brought to our attention as

275

276 LECTURE XIII.

apparently combined with albumin. Nucleoproteids have, however, been
obtained by salting them out. Although we have no doubt that such
compounds with nucleic acid exist, especially of the basic proteins, like
the histones and protamines, still we must admit that no convincing proof
has yet been presented that there is such a state of combination in the cells
themselves. We are accustomed to look upon substances which we always
find in given localities, and are never absent, as being particularly important
for the functions of the cells and tissues, especially when we find these in
the parts of cells to which we assign great importance. Although such an
assumption is probably true, we should be concealing the actual state of
our knowledge, if we failed to mention the fact that the exact significance
of the nucleoproteids is still unknown to us, and that we do not, at present,
understand their relations in cell-metabolism.

We have already stated that the nucleoproteids are composed of two
constituents, one of which is an albumin, and the other nucleic acid. It
is not yet clear to us how we shall conceive the formation of the proteids
from these two components. It has been shown that the decomposition
into albumin and nucleic acid portions does not always take place as if it
were a simple process. We obtain the impression that the nucleic acid is
united with two parts of albumin. One part can be easily split off, the
other with much more difiiculty. The following scheme expresses this
conception:

Nucleoproteids
/\
Albumin Nuclein
/\
Albumin Nucleic acid.

When albumin is split off from the nucleoproteid, a part of the protein
remains combined with the nucleic acid. This product is called nuclein.
This was first observed by Miescher on digesting a nucleoproteid with
pepsin and hydrochloric acid. The albuminous portion, which is most
easily split off, is decomposed, while the nuclein precipitates. More recent
investigations have shown that active pepsin may even disintegrate the
nuclein, thus leaving the pure nucleic acids behind.

We are especially interested here in the nucleic acids. We have already
considered the protein constituent as far as it is known. All the nucleic
acids contain phosphorus. When decomposed, they produce phosphoric
acid and nuclein bases. Other products are also formed when the nucleic
acids are decomposed. A carbohydrate group has been split off in some
cases; while from others pyrimidine groups have been obtained. We shall
here first consider all of the known cleavage-products of the different
nucleic acids, and not pay any attention at present to the composition of
these different acids. All of these nucleic acids which have been studied,

THE NUCLEOPROTEIDS. 277

except inosie acid (from extract of beef) which forms crystalline salts/
are amorphous and react acid. They are easily dissolved in water con-
taining ammonia or alkali, and form insoluble salts with the heavy metals.

Phosphoric acid is, as we have said, quite generally found among the
cleavage-products of the nucleic acids. We do not know how it is united
in the molecule. The occurrence of representatives of the group of purine
bases is especially important. They vary according to their origin, and
the number of bases participating in the constitution of the nucleic acids
is also a variable one. J. Piccard - early met with these compounds in his
investigations of the nucleins. The numerous observations of A. Kossel '
have indicated their wide distribution, and also the nature of the purine
bases present.

We wish to state in advance that the purine bases are very closely related
to an important metabolic end-product, uric acid, not only from a purely
chemical point of view, but also because recent experiments have indicated
intimate biological connections. We wish, therefore, to describe briefly
the most important points with regard to the constitution of this class of
bodies. It will then be easier for us to follow the individual purine bases
in their course through the organism, and to judge of the part they play in
metabolism.

Uric acid, the earliest known member of this series, was discovered in
urine and bladder stones, as far back as 1776, by Scheele^ and Bergmann.^
We will add that Pearson" has shown the presence of uric acid in "chalk-
stones," and that Fourcroy and Vauquelin ' shortly after proved it to be an
essential constituent of the excrement of birds. Finally William Prout,*
in 1815, found that the excrement of the boa-constrictor contained as.
much as 90 per cent of uric acid.

Uric acid was thoroughly investigated by Liebig. A large number of
its important decomposition products also became known, without, how-
ever, indicating the true constitution of the acid itself. Wcihler and Liebig *
mention its close relation to allantoine , even then regarded as a component
of the allantoic fluid. They obtained urea and alloxan by treating uric acid

' J. von Siebig: Ann. 62, 317 (1847) and Haiser: Monatsh. 16, 190 (1895).

' J. Piccard: Ber. 7, 17U (1874).

' A. Kossel: Z. physiol. Chem. 4, 290 (1880); ihid. 7, 7 (1882); ibiV/. 10, 248 (1886);
12, 241 (1888).

* K. W. Scheele: Examen chimicum Calculi urinarii, Opuscula II, 73 (1876).

' T. Bergmann: Opuscula IV, 232 (1876). Cf. E. Fischer: Ber. 32, 4.35 (1899). Cf.
also Synthesen in der Purin- und Zuckergruppe, F. Vieweg & Sohn, Braunschweig,
1903, and Untersuchungen in die Puringruppe (1882-1906), J. Springer, Berlin, 1907.
K. Bunte: Inaug. Diss. Berlin, 1905.

« Pearson: Phil. Trans, of the Royal Soc. London, 15, 1798.

' Ann. de chim. 56, 258 (1905).

' Ann. Phil. 6, 413 (1815).

» Ann. 26, 241 (1838).

278 LECTURE XIII.

with nitric acid. From alloxan they obtained a large number of closely
related compounds. We are indebted to Adolf Baeyer ' for establishing
the constitution of alloxan and its closely allied derivatives. We will also
mention the discovery of A. Strecker,- that heating uric acid with concen-
trated hydrochloric acid in a sealed tube to 170 degrees, produces glycocoll,
carbon dioxide, and ammonia, water entering into the reaction:

C5H4N4O3 + 5 H2O = CH2 . (NH2) . COOH + 3 CO2 + 3 NH3.
Uric acid Glycocoll

From this fact, Strecker regarded uric acid as a glycocoll united with
cyanic acid, and assumed that the uric acid first broke down into glycocoll
and cyanic acid; the latter then being further decomposed into carbon
dioxide and ammonia:

C5H4N4O3 = CH2 . (NH2) . COOH + 3 HCNO.

This decomposition of uric acid was of great significance, because, using
it as a basis, Horbaczewski ' next produced uric acid by fusing glycocoll
with urea at 220°-230° C:

/NH,

3 CO + CH2 . NH2 . COOH = C5H4N4O3 + 3 NHs + 2 H^O.
\NH,

On heating urea, ammonia is set free, cyanic acid also being formed,
which can then act further on the glycocoll. Another synthesis was accom-
plished by melting urea with tri-chlor-lactamid:

/NH2

2 CO +C3CI3O2H0 . NH2 = H20 + NH4Cl + 2 HCI + CSH4N4O3.
\NH2

These syntheses did not lead to an exact conception of the constitution of
uric acid. It was only through the carefully-planned, sj'stematic inves-
tigations of Emil Fischer, that light was suddenly thrown on the whole
group of the purines and their derivatives. Not only does the entire chem-
istry of all the compounds of this group depend on his work, but also all
biological research in this field.

We cannot at this place trace the development of all of Emil Fischer's
work, but will merely single out the points which are most important in
our study .^ It is necessary in the first place to establish the relations
which exist between the various members of this group. Emil Fischer
based his work upon the relations of all the members of this group to
purine. He finally succeeded in obtaining purine itself, thus laying the

' Cf. his complete works, F. Vieweg & Sohn, Braunschweig, 1906, vol. i, p. 57.

2 Ann. 146, 142 (186.S).

' Monatsh. 3, 796 (1SS2); 6, .356 (1885).

* Cf. E. Fischer: Ber. 30, 549, 18.39, 2226 (1897), and 31, 104 (1898).

THE NUCLEOPROTEIDS 279

cornerstone for his whole investigation of the uric acid group. Purine is
a strong base, readily soluble in water. Its constitution is evident from
its preparation. It has the following structure:
N = CH

I I

HC C— NH

II II NpH

Purine

In order to make it possible to have a uniform nomenclature for the
numerous representatives of this group, Emil Fischer numbered the purine
ring in the following manner:

N,=C6

I I

C2 C5-N7\

II II , Cs
N3— C4-N9^

We shall make use of this scheme.

Uric acid itself has the following structural formula:

HN— CO

I I
COC— NH^

I II )co

HN— C— NH •^

Uric acid (2, 6, 8-trioxypurine)

This formula harmonizes with the following important transformations
of uric acid.

By heating with hydriodic acid and fuming hydrochloric acid in a sealed
tube, glycocoll, carbon dioxide, and ammonia are produced.

Oxidizing with nitric acid or chlorine produces alloxan and urea.
Alloxan is mesoxalyl urea:

HN— CO NH— CO .TTi

I I I I ^^^'

CO C— NH CO CO + CO

I II )C0 + 0 + H20 = I I Xj^jj

HN— C— NH^ NH— CO '^"2

Uric acid Alloxan

By oxidation of alloxan we obtain parabanic acid, which is oxalyl urea:

CO— NH

I /^^

CO— NH

Parabanic acid

which decomposes, on boiling with water, into urea and oxalic acid.

280

LECTURE XIII.

The transformation of uric acid into aUantohic liy oxidation is very

important :

NH— CH— NH ,

co; I ;co.

^NH— CO NH/
Allantoine

There are a large number of compounds important to the biologist which
are very closely related to uric acid. We have mentioned that the nucleic
acids, when decomposed, produced purine bases; in fact, the following:
xanthine, hi/po-xanthine, adenine, and guanine. Their structural formulae
are as follows:

HN— CO

HN— CO

CO C— NH CO C— NH ^

I I )co I II V;h

HN— C— NH^ HN— C— N "^

Uric acid = Xanthine =

2, 6, 8-trioxypurine 2, 6-dioxypurine

HN— CO

I I

HC C— NH ,

II II
N— C— N

Hypoxanthine
6-oxypurine

^CH

NH— c;o

NH. . C C

-NH

— N'

CH

Guanine =
2-amino-6-oxypurine

N = C— NHj

I I

HC C— NH,

II II
N— C— N '-^

Adenine =
6-aminopurine

•CH

In this connection we will add that substances closely related to the
purines have also been separated from the vegetable kingdom. These are
caffeine, theobromine, and theophylline. The first two are found in table
acees.sories, caffeine being present in coffee and tea, while theobromine is
a constituent of cocoa. The relation of these three compounds to one
another is indicated by their formulae:

CH3 . N— CO

I I

CO c— n:

CH,

HN— CO

CO c— n;

.CH3

r^

CH

r^CH

CH3.N— C;— N'-' CH3.N— C— N^

Caffeine = Theobromine =

1, 3, 7-trimethyl-2, 6-dioxypurine 3, 7-dimethyl-2, 6-dioxypurine

CH3 . N— CO

I II
CO C— NH

I " CH

CH3 .N— C— N.^^"

Theophylline =

1, 3-dimethyl-2, 6-dioxypurine

THE NUCLEOPROTEIDS. 281

Very closely related to this group of nucleic acid cleavage-products are
other compounds, which, instead of having a purine ring, contain one of
pyrimidine:

(1) N = CH (6)

I I

(2) HC CH (5)

II II

(3) N— CH (4)
Pyrimidine

The syntheses of the members of this series and their constitution indi-
cate their close relation to the purine derivatives. We are mainly indebted
to A. Kossel for their discovery. Ascoli ' first found uracijl in yeast-
nucleic acid. It has the following constitution:

NH— CO

I I

CO CH

I II

NH— CH
Uracyl =
2, 6-dioxypyrimidine

Emil Fischer and Georg Roeder - succeeded in synthesizing it. These
same authors also established the constitution of another pyrimidine base,
thymine. It is a 5-methyluracyl,

NH— CO

I I

CO C . CH3

I II

NH— CH

Thymine =

5-methyluracyl (5-methyl-2, 6-dioxypyrimidine)

This compound was first isolated from thymus nucleic acid by A. Kossel
and Neumann.^

Finally, we are acquainted with a third pyrimidine derivative, cytosine,
which was also separated from thymus nucleic acid by Kossel and
Neumann.'' It has been synthetically prepared by Wheeler and Johnson,^

' A. Ascoli: Z. physiol. Chem. 31, 161 (1900-01). A. Kossel and H. Steudel: ibid.
37, 245 (1902).

' E. Fischer and G. Roeder: Ber. 34, 3752 (1901).

' A. Kossel and A. Neumann: ibid. 26, 275.3 (1893); Z. physiol. Chem. 22, 188 (1896).
H. Steudel and A. Kossel: ibid. 29, 303 (1900). H. Steudel: ibid. 30, 539 (1900); 32,
241 (1901). W. Jones: ibid.. 29, 20 (1899); 30, 461 (1900). W. Gulewitsch: ibid. 27,
292 (1899); 27, 368 (1899). O. Gerngross: Ber. 38, 3408 (1905).

* A. Kossel and A. Neumann: Ber. 27, 2215 (1894). a'. Kossel and H. Steudel:
Z. physiol. Chem. 37, 177 (1902); 37, 377 (1903); 38, 49 (1903).

' H. L. Wheeler and T. B. Johnson: Am. Chem. J. 29, 492 and 505 (1903).

282 LECTURE XIII.

and has the following structural formula:

HN - C . NH2

I II

CO CH

I I

N = CH
Cytosine = 6-amino-2-oxypyrimidine

Carbohydrates — • in fact mostly pentoses — have also been obtained
from the nucleic acids in conjunction with the purine, and pyrimidine bases,
and phosphoric acid. Such a typical pentose is xylose. Yeast nucleic acid
is supposed to contain an hexose. It is also assumed that an hexose par-
ticipates in the constitution of the thymus nucleic acid. Lsevulic acid
is obtained therefrom by an energetic decomposition.

It has recently become questionable whether all of the above com-
pounds are to be regarded as primary cleavage-products of the nucleic
acids. Steudel ' has shown it to be probably true that adenine and guanine
are the only primary building stones of the purine bases, and thymine
and cytosine of the pyrimidine bases. Hypoxanthine, xanthine, and
uracyl are formed secondarily by oxidation in the breaking down of the
nucleic acids. This discovery lessens the value of the numerous inves-
tigations concerning the purine and pyrimidine bases in the different
nucleic acids. It also explains why different authors have obtained
divergent results in the study of nucleic acids from the same source.
Although this indicates a great gap in our knowledge concerning the
amount of individual building stones present in the nucleic acids, which
can be filled only by the assumption of secondary transformations, stili
on the other hand, it is very good news to find that Neuberg and Brahn^
and Bauer' have succeeded in clearing up the constitution of inosic acid.
From the latter, one molecule each of hypoxanthine, phosphoric acid,
and xylose or arabinose is always obtained. It is at present an open
question whether the purine bases here observed are to be regarded as
of primary or secondary formation, and whether perhaps adenine is not
here also the primary building stone. The question that next arises is
with regard to the way the components of the nucleic acids are held
together in the molecule. Undoubtedly this again will only be answered
when synthesis has established the relations.

Burian^ attempted to decide how the purine bases are held in the
nucleic acid molecules. In one case he based his observations upon the
fact that the purine bases, unlike the other components of the nucleic

' H. Steudel: Z. physiol. Chem. 49, 406 (1906).

' C. Neuberg and B. Brahn: Biochem. Z. 5, 4.38 (1907).

» Friedrich Bauer: Beitr. chem. Physiol. Path. 10, 345 (1907).

* R. Burian: Ber. 37, 696 and 708 (i904) ; Z. physiol. Chem. 42, 297 (1904).

THE NUCLEOPROTEIDS. 283

acid molecule, are very easily split off. Purine bases can be detected even.
when the material is merely dissolved in water at 60 degrees. Boiling
with water for ten minutes is sufficient to separate practically all the
purine bases. We are justified in looking upon the purines as being
primary cleavage-products of the nucleic acid molecule, rather than
resulting from secondary causes. We may consider them as being com-
posed of a condensed nucleus, consisting of a pyrimidine and an imidazole
ring, as may be seen by the following structural formulae:

(1) N=(6)CH (1) N-(6)CH

II II

(2) HC (5) C— (7) NH (2) HC (5) CH (a)HC— NH(n)

II' II i^^^^^ II II II )CH(m).

(3) N— (4)C— (9)N (3) N — (4) CH (/3)HC— N

Purine Pyrimidine Imidazole

Corresponding to this assumption, the purines show reactions, which are
characteristic of one or the other component. Imidazole possesses the
property of reacting with diazo-benzene chloride, forming a red, crystalline
product, which is (n) diazo-benzene-imidazole. This reaction also applies
to those imidazoles whose a, /?, or ju. positions have been substituted; but
not to those in which the n-position is substituted. The purines act in
a very analogous manner. Substitution in the pyrimidine ring is without
influence on the appearance of this reaction. On the other hand, the re-
action is negative when the imide hydrogen of the imidazole ring, in position
7, is replaced. The reaction is positive in xanthine, hypoxanthine, guanine,
adenine, theophylline; it fails with theobromine (3, 7-di-methyl-xanthine)
and with caffeine (1 , 3, 7-tri-methyl-xanthine) . The nucleic acids, however,
even if liberally endowed with bases, do not react with diazo-benzene-
sulphonic acid. This last reaction only takes place when purines are being
split off at the same time. Taking the above facts into consideration,
we arrive at the conclusion that the purine bases are united with the nucleic
acid residue through its nitrogen atom in the seventh position. Another
series of observations indicate that the purine bases are linked in the first
place to the phosphoric acid portion. It is a very striking fact that those
purine bases, which are so easily split off by boiling water, are only released
with great difficulty by boiling with sodium hydroxide. Certain organic
phosphoric acid amides behave similarly. Burian believes that guanine is
linked in the nucleic acid molecule in the following way:

HN— CO P =

I I /
NH2 . C C— N ^

II II ^CH
N— C— N^

2S4 LECTURE XIII.

The proof that such a state of combination exists in the nucleic acid
molecule is not satisfactory. Other possibilities exist. Burian has, never-
theless, shown that such a linkage is probable.

In discussing the proteins, we found that a knowledge of the amino
acids participating in the constitution of the individual albumins often
gave us valuable suggestions regarding their behavior in the animal
organism. The relations of most nucleic acids, on the other hand, are
as we have already stated, not so clear, because 'they have not all
been studied in the same way, nor with the same care. Above all we
have no means for deciding which cleavage-products are to be regarded
in any given case as primary or secondary. Inosic acid is an excep-
tion, as its composition has been established, and we are justified in con-
sidering it as a simple substance. Perhaps this is also true of guanylic
acid ' isolated by Bang and Raashon ^ from the pancreas. In the break-
ing down of this nucleic acid, guanine is the only purine base that could
be detected in the presence of phosphoric acid and a carbohydrate. As
to whether the remaining nucleic acids are simple substances or mixtures,
we have no means of knowing. It will be best here to mention merely
the most important nucleic acids. They are almost always designated by
the name of the organs from which they were obtained.

The nucleic acids from the spermatozoa have been longest known.
Since F. Miescher ^ first called attention to them, they have been the subject
of frequent investigation.^ It appears that the nucleic acids obtained
from the various kinds of spermatozoa are closely related to one another.
Certain observations indicate a far-reaching similarity. Great care should
be taken, however, in drawing any conclusions regarding the identity of
'the various nucleic acids from analytical values or knowledge of the
cleavage-products. According to the grouping of the cleavage-products,
many differences may appear which are as yet hidden from our view. It is
noteworthy that the percentage of phosphorus present in the nucleic acids
isolated from the cells of ripe semen is, in general, a very constant one.
The values range between 9.11 per cent and 9.62 per cent. Salmon
nucleic acid, according to Schmiedeberg,^ has the following composition:
C, 37.42 per cent; H, 4.19 per cent; N, 15.24 per cent; and P, 9.64. AD
the known purine bases have been obtained in the cleavage of the
spermatozoa nucleic acids. The values are given on the following
page.

1 I. Bang Z. physiol. Chem. 26, 133 (1898-99); 31, 241 (1900-01).
^ I. Bang and C. A. Raaschon: Hofmeister's Beitr. 4, 175 (1903).
' F. Miescher: Verb, naturf. Ge.sell. Basel, 6, 1.38 (1874). Cf. also the complete works
of F. Miescher: loc. cit. 2, 55; Arch. e.\per. Path. Pharm. 37, 100 (1896).
* R. Burian: Ergeb. Physiol. 3, 1, 48 (1904).
» O. Schmiedeberg: Arch, exper. Path. Pharm. 43, 57 (1900).

THE NUCLEOPROTEIDS.

285

100 g. Dry Substance Contains

Adenine. | Guanine. Hypoxanthine. Xanthine,

Nucleic acid from the testes of the bull'

Spermatozoa of the bull

Spermatozoa from the testes of a boar'

Spermatozoa of the carp ^

Salmon nucleic acid. 1st preparation' .
Salmon nucleic acid. 2d preparation
Pancreas '

0.736
0.126
1.181
0.360

0.248
0.187

0.127
0.193

1.962
0.207
0.635
0.309
0.664
1.208
0.154

6.039
0.352
2.057
2.278
2.924
3.914
0.740

We must once more state that too much reliance should not be placed
upon these figures. Burian and Walker HalP called attention to the fact
that, in the preparation of the purine bases, oxypurines (xanthine and
hypoxanthine) might be formed from the aminopurines (adenine and
guanine). As we have seen, Steudel has proved this directly. This
probably accounts for the fact that Schmiedeberg, in investigating salmon
nucleic acid, found guanine and adenine, but no xanthine nor hypo-
xanthin.

Thyvio-nudeic acid from the thymus gland is another nucleic acid which
has been investigated considerably.* Phosphoric acid, thymine, cytosine,
guanine, adenine, Isevulic acid, and ammonia were obtained by hydrolytic
decomposition. Guan)jlic acid, obtained from the pancreas, broke down
into guanine, phosphoric acid, and a pentose. A nucleic acid has also
been isolated from the spleen. We may add, finally, that analogous
products can also be obtained from the plants. Triticonucleic ^ acid',
obtained from the embryo of wheat, has been most studied. It yields
by hydrolysis : guanine, adenine, cytosine, uracyl, phosphoric acid, and a
pentose. Nucleic acids have also been isolated from tubercle-bacilli and
from yeast.

We cannot at present say anything regarding the nucleic acids and their
cleavage-products, and shall confine ourselves in the following to the dis-
cussion of their participation in metaboUc processes. We will state, at
the start, that there can no longer be any doubt that the nucleo-
proteids are disintegrated in the cell-metabolism, and participate to the
same extent in the reconstruction. There is scarcely any question but
that the animal cell obtains the components of the nucleoproteids already
formed in the food. It does not seem probable that the purine and pyri-

• Y. Inoko: Z. physiol. Cheni. 18, 57 (1894).

' S. Schindler: ibid. 13, 432 (18S9).

' R. Burian and W. Hall: ibid. 38, .366 (1903).

' H. Steudel: ibid. 42, 165 (1904); 43, 402 (1904); 46, 332 (1905); P. A. Levene, 32,

541 (1901); 37, 402 (1902-03); 38, 80 (1903); 39, 4 (1903); 39, 479 (1903); 43, 199
(1904); 45, 370 (1905); .A.m. J. Physiol. 12, 213 (1905).

^ T. B. Osborne and J. F. Harris: Z. physiol. Chem. 36, 85 (1902).

286 LECTURE XIII.

midine bases are synthetically formed by the cells for this purpose. To be
sure, the animal organism — at least, that of the birds and the reptiles — is
capable of building up uric acid. From the whole manner of its formation
it would seem very improbable that the purine bases have the same origin.
In this connection we must indeed recall the work of F. Knoop and
A. Windaus,' from which we can easily assume a synthetic production of a
compound which is important to the cell-metabolism. These two inves-
tigators have shown that the action of ammoniacal-zinc-hydroxide on
grape-sugar in the cold produces an oxygen-free base in large quantity;
namely, methyl-imidazole :

CH3— C— NH

I >H
CH-N

This gives us a connecting link between the carbohydrates and the
purine bases. It is possible that analogous reactions may occur in the
plant organism. The animal cell would hardly look to the carbohydrates
as a source for the production of nitrogenous material; at least, nothing
at present known would indicate that it does. We will state here that,
in spite of the large number of recent observations in the field of purine
metabolism, it is still impossible for us to give an exact account of the
influence of this class of bodies on the total metabolism, and even less so
in the case of the nucleoproteids and nucleic acids in the individual cells.
We know that the animal organism utilizes materials containing purines
for its requirements. There are nutrient substances, like meat, rich in
purine bases, and others, again, containing less of these. Milk belongs to
the latter class. The animal cell undoubtedly requires nucleic acid and
also purine bases for the purpose of building up nucleoproteids. They con-
stantly decompose — as we shall soon see — those constituents which contain
purine and replace them again. From this we can easily imagine that the
cell utilizes the nucleic acids either in their original or converted form to
replace wasted material or to construct new cells. We shall see later that
the animal organism continues to break down material containing purines,
even in cases of extreme starvation, some of the derivatives formed by
this process appearing in the urine. There is a far-reaching analogy here
to the behavior of the proteins in the organism. It is possible — in fact
very probable — that the nucleoproteids in the form of nucleic acids are
utilized by the cells, at least in part, in the manner just indicated. We
must not forget, however, that we have no absolute proof of this. In fact
there are some observations which point to the probability that the animal
organism, like the plants, is also capable of directly synthesizing the purine

' F. Knoop and A. Windaus: Ber. 36, 1166 (1905). Hofmeister's Beitr. 6, 392 (1905).

THE NUCLEOPROTEIDS. 287

bases. Tichomiroff ' has shown that hibernating insect eggs contain only
traces of purine bases, while the maturing eggs show a much larger per-
centage of these. A. Kossel ^ finally showed that the yolks of unincubated
eggs contained practically no purine bases. After fifteen days' incubation
larger amounts of guanine and hypoxanthine could be detected. We must
also refer to the work of Burian and Schur.' They estimated the amounts
of bases present in new-born animals, and compared these values with
those obtained from older sucklings. Although the latter had received
almost no purine bases with their nourishment, the milk, the amount of
these substances continually increased. Finally, we must remember the
work of F. Miescher,* who found that even the salmon, during the fasting
period when it remains in fresh water, not only builds up nucleins from the
simplest components, but newly forms the purine bases as well. All these
important observations show us what difficult syntheses the animal cell is
capable of effecting. There does not seem to be any reason for doubting
that the most varied constituents of the tissues of the animal organism are
built up of the simplest components. It is still an open question regarding
the manner in which the growing organism carries out this process.

It is, at present, impossible to make any definite statements concerning
the products utilized in the synthesis of the purine bases. It is indeed
possible that further investigations with histidine and its behavior in the
animal organism may give us some clew to this process. This albuminous
cleavage-product, as we have seen, is very probably an a-amino-/?-imid-
azole-propionic acid:

CH— NH

II >H

C — N

CH,

I
CH . NH2

I
COOH

If this be true, we have another bridge from the proteins to the purines.

We specifically call attention to our ignorance of the relations of the
nucleic acids to the general metabolism, because recent investigations
on the disintegration of these substances in the tissues have indicated
the ease with which these gaps in our knowledge may be overlooked.
They immediately become apparent when we attempt to explain the

■ A. Tichomiroff: Z. physiol. Chem. 9, 518 (1885).

» A. Kossel: Z. physiol. Chem. 10, 24S (1886).

' R. Burian and H. Schur: ihid. 23, 55 (1897).

* F. Miescher: Arch, exper. Path. Pharm. 37, 100 (1896).

288 LECTURE XIII.

causes of the well-known metabolic derangement, gout, which is un-
doubtedly closely related to a disturbance of the purine assimilation.
Our uncertainty begins when we proceed to follow the behavior of the
nucleic acids in the alimentary tract, although we are a little better informed
regarding the decomposition of the nucleoproteids themselves. The latter
are, at the start, vigorously attacked by pepsin and hydrochloric acid
in the stomach. The loosely-linked albuminous component is split off
and converted into peptones. Nuclein then separates in an insoluble
form, but later on is partially dissolved. Trypsin likewise separates
the albuminous component from the nucleoproteids. The nucleic acids,
however, seem to remain entirely unaltered. They must, therefore,
occupy a class by themselves among the food materials, because all the
others so far discussed are largely disintegrated in the intestine in order
to supply the tissue cells, and partly those of the intestine itself, with
the necessary material for their individual needs. We would expect,
a priori, that the nucleic acids would also have to be disintegrated to
make them available. Up to the pre.sent time but one ferment has been
isolated from the ti.ssues capable of separating nucleic acids into their
components. This is the so-called nuclease.^ Trypsin destroys this
ferment. Nuclease has been found in the pancreas of the dog and the
thymus gland of the calf. Undoubtedlj', such ferments must be widely
distributed in the tissues. They account for the first stages of the cleavage
and degradation of the nucleic acids. Recent investigations ^ have shown
that neither the active nor the inactive pancreatic juice is able to decom-
pose the nucleic acids into their components; both, however, are capable
of so altering them that their entire characteristics are changed, thus
making them more easily dialyzable. Even the cell walls of the intestine
possess ferments which are capable of completely decomposing the altered
nucleic acids. The animal organism evidently treats this valuable material
in a very economical manner. The nucleic acid cleavage-products are
difficultly soluble in water, and not easily absorbed, as feeding experi-
ments with purine bases have proved. These experiments indicate that
the complete disintegration of these compounds takes place only in the
walls of the intestine. Material, foreign to the organism, is there pre-
pared for its requirements. We are unacquainted with the exact manner
in which the pancreatic juice changes the nucleic acids. It may possibly
be that it acts as the beginning of a hydrolytic decomposition. The
disintegration proceeds in stages, and we must expect to meet complexes
analogous to the peptones, and dextrins. We have not the least doubt
but that the nucleic acids very closely resemble the albumins in their
entire construction and the way they are broken down.

' F. Sachs: Z. physiol. Chem. 46, 337 (1905).

' E. Abderhalden and A. Schittenhelm: ibid. 47 (1906).

THE NUCLEOPROTEIDS. 289

A part of tne nucleic acid of the food is undoubtedly decomposed by-
bacteria in the intestines. Purine bases are present in the faeces.' Martin
Kriiger and Schittenhelm ^ have shown that only a small part of the purine
bases in the faeces could originate in this manner. It has been found that
the quantitive distribution of the various purine bases in the faeces corre-
sponds very closely to that in the different organs, and that the main source
of supply is undoubtedly the degenerating intestinal epithelium and dead
bacteria. Only small amounts of bases are introduced into the intestines
by means of the pancreatic and intestinal juices.

Until recently, we knew but very little about the relation of the nucleo-
proteids, or, rather, of the nucleic acids and their cleavage-products, to the
decomposition products of the general metabolism. Indeed, many scien-
tists, largely from purely chemical considerations, were strongly in favor
of assigning to the nucleins a relation to the formation of uric acid. It
remained, however, for recent experiments to show that in man, and
mammals in general, the greater part and perhaps all of the uric acid
results from the decomposition of the nucleic acids and their cleavage-
products, e-specially the purine bases.

For a long time the attempt had been made to show that uric acid was
the antecedent of urea in the breaking down of proteins. In fact, the
amount of uric acid in the urine was even considered to be a direct expres-
sion of the activity of oxidations in the animal organism. The more
extensive these oxidation processes were, the less uric acid would be found
in the urine. Evidence against this assumption was brought forward from
time to time, and, above all, it was always claimed that in no case could
an}' direct relationship be shown between the uric acid excreted and the
disintegration of the albumins, and that there was no evidence that it
indicated the extent of the oxidation processes. Scientists always came
back to the above view, however, because it was not found possible to
show positivel}' that there was an increased elimination of uric acid after
the administration of nucleic acids and purine bases. It was only by the
experiments of Horbaczewski ' that the problem was cleared up.

Horbaczewski showed with mammals that if the pulp or extract of
organs were digested for several hours out of contact with the air, purine
bases were formed, while exposure to the air gave rise to uric acid. On
adding nucleoproteids a better yield of uric acid was obtained. It was

' A. Schittenhelm and F. Schroter: Z. phy.sioI. Chem. 39, 203 (1903). A. Schitten-
helm and C. ToUens: Z. innere Med. 25, No. 30 (1904).

■ M. Kruger and A. Schittenhelm: Z. physiol. Chem. 45, 14 (1905); 35, 153 (1902).
A. Schittenhelm: Dent. Arch. klin. Med. 81, 423 (1904).

' Horbaczewski: Monatsh. 10, 624 (1889); 12, 221 (1891). P.Giacosa: Att. R. Ace.
Scienze di Torino, 25, 726 (1891). W.Spitzer: Pflijger's Arch. 76, 192 (1899). H.Wiener:
Verh. xvil, Kong, innere Med. 1889; 622 Arch, exper. Path. Pharm. 42, 375 (1899).

290 LECTURE XIII.

also shown by feeding experiments that the administration of niicleo-
proteids and of purine bases increased the eHmination of uric acid. Sim-
ilarly the formation of uric acid is increased when a food rich in purine
bases — e.g., meat, liver, thymus, etc. — is added to a definite diet.'

Horbaczewski himself believed that the uric acid was derived in the
first place from the nucleic acids, or their purine bases, obtained from
leucocytes, and his work was followed by a large number of investigations
concerning this question. It was, in fact, found that there was a certain
relation between the amount of uric acid eliminated and the number of
leucocytes. A particularly pregnant example, according to this view, is
given in leucaemia, a disease which in its entire aetiology is but little under-
stood. One of its most prominent symptoms is a more or less extensive
increase in the number of leucocytes. The observation that the increased
elimination of uric acid, so often noted during this disease, is dependent
on the destruction of leucocytes, is undoubtedly true. The generalization
that the uric acid of urine could only result from the destruction of the
above-mentioned cells was not, however, correct.^ All the other cells of
the organism must be considered in the same manner. The most impor-
tant result of the investigations of Horbaczewski is that the purine bases,
in minced organs and tissue extracts, can, in the presence of oxygen, be
converted into uric acid.

To prevent any misunderstanding, we will state at this point, that the
increased elimination of uric acid can, in no case, be looked upon as evidence
of an increased cell destruction. It may just as easily arise from an
increased cellular metabolism; i.e., from the breaking down and recon-
struction of the cell-body, and especially of the nuclei.

Before discussing the mechanism of the conversion of purine bases
into uric acid, we wish to devote a little space to an important investiga-
tion of Burian and Schur.' These two scientists showed that by a diet
containing no purine bases it is possible to diminish appreciably the excre-
tion of uric acid, but not to prevent it entirely. It is noteworthy that the
amounts of uric acid then excreted remain practically constant for each
individual, but vary with different individuals.* Burian and Schur desig-

' R. Burian: Med. Klin. 1, 131 (1905). E. Salkowski: Virehow's Arch. 117, 570
(1889). C. V. Noorden: Lehrbuch d. Path. d. Stoffwechsels, 54, Berlin, Hirshwald,
1893 (new ed. 1906). C. Dapper: Berliner Klin. Wochsch. 30,619 (1893). W. Cam-
merer: Z. Biol. 28, 72 (1891); Z. physiol. C'hem. 33, 139 (1896). A. Schittenhelm:
Z. Stoffw. u. Verdauungskrankheiten, 5, 226 (1904). H. Wiener: Ergebnisse Physiol.
I, 1, 555 (1902).

' Mares : Monatsh. 13, 101 (1892).

' R. Burian and H. Schur: Pfliiger's Arch. 80,241 (1900); 87,2.39(1901). Cf. E.W.
Rockwood: Am. J. Physiol. 12, 38 (1905).

« Schreiber and Waldvogel: Arch, exper. Path. Pharm. 32, 69 (1899). Cf. M. Kauf-
mann and L. Mohr: Arch. klin. Med. 74, 141 (1902).

THE NUCLEOPROTEIDS. 291

nate the amount, of uric acid eliminated with a diet free from purine bases
as endogenous uric acid, and contrast this part of the total uric acid elimina-
tion during an ordinary diet with that obtained from the purines in the
food. The amount of the latter, which they designate as exogenous uric
acid, naturally varies, and is dependent upon the quantity of purine
bases ingested and absorbed. The amount of endogenous uric acid
remains constant, even from a diet rich in purine bases. The uric acid
arising from the purine bases is added to that of endogenous origin.
Burian states that the amount of endogenous uric acid excreted daily by
a normal adult ranges between 0.3-0.6 gram. The endogenous uric
acid value is a direct expression of the extent of cell activity, which, as we
know from various observations, is very carefully regulated and adjusted
for each individual.

Although we may look upon this conception of endogenous and exoge-
nous uric acid, in the sense suggested by Burian and Schur, as a distinct
advance in our knowledge of the course of cell-metabolism, we must,
nevertheless, emphasize the fact that this separation between the two
sources is merely a superficial one. In no case are we justified in con-
cluding that the total purine metabolism is sharply divided into two
phases; i.e., that the purines in the food are immediately converted into
uric acid, nor that cell-metabolism, in the narrowest sense of the word,
takes place independently. We have no doubt that the purine bases,
and the other components of nucleic acid, replace continually material
used in the building up of cells, and particularly their nuclei, and thus
take part in the formation of endogenous uric acid, of a later period.
Although we know that the total nitrogen of the food, as a rule, soon re-
appears in the urine, we nevertheless assume that the albumins, at least
to some extent, participate in cell-metabolism, and in the construction of
cells partly supplying material, and partly acting as a source of energy.
Similarl}', the purine bases in the food undoubtedly participate in cell-
metabolism. The endogenous purine value maj- perhaps be compared,
within certain limits, with that amount of albumin which the cells require
even during starvation. We do not mean to represent that this compari-
son is an absolute one. It merely suggests the similarity between the
metabolism of albumin and purine — both are very closely related to
cellular metabolism. We must be especially cautious not to differentiate
sharply the endogenous and exogenous uric acid with regard to the total
purine metabolism of the cells.

The question arises, What sources have we to assume in particular for
the endogenous uric acid? In general it may be said that the endogenous
uric acid may be traced primarily to the decomposed nuclear material,
and also to the cells which have been completely destroyed. R. Burian '

» Z. physiol. Chem. 43, 494 (1905).

292 LECTURE XIII.

has recently called our attention to the importance of considering the
muscles as a source of purine bases, and, consequently, they are related
to the formation of uric acid. He showed in the first place that the endo-
genous uric acid elimination in human beings during twenty-four hours
was not appreciably influenced by muscular activity, whereas hourly
values were distinctly changed. Vigorous muscular exertion is followed
by an hour of increased ehmination of urinary purine. This increase,
during the time of work, does not influence the uric acid, as such, but
principally the purines. The noticeable increase in uric acid elimination
is only evident some time later. By a subsequent diminution of the uric
acid and purine eliminations, the daily uric acid and purine values are
practically unaffected by periods of rest or of activity. Naturally such
investigations can be carried out only when no food is eaten, or at least
none containing purine, or the amount of purine bases in the food must be
definitely known. Burian finally, in order to establish more closely the
relations of the muscles to purine metabolism, caused blood to flow through
the surviving muscles of a dog. It was found first of all that the liquid
which was originally perfectly free from uric acid always contained it after
a short time. If the muscle was stimulated, purine bases appeared in
considerable quantity, and chiefly hypoxanthine. The discovery is also
very important that the amount of hypoxanthine in the muscle itself is
greatly increased when it is tetanized. From this we must assume that
the muscle, when at re.st, constantly oxidizes hypoxanthine to uric acid.
When its metabolism is increased by gi-eater demands upon it, the muscle
cells are no longer able to oxidize all of the purine bases, and especially
the hypoxanthine, so that then unchanged hypoxanthine is given up to
the blood. It is very important that according to these observations the
muscle cells are constantly forming hypoxanthine. These investiga-
tions are not to be regarded, however, as perfectly conclusive. We have
mentioned them here because from them we may perhaps expect to obtain
the first explanation of the part played by the purines of the food in
cellular metabolism, and concerning the extent of the synthetic formation
of purine bases.

We have been informed recently concerning the breaking down of the
individual purine bases to uric acid, and their subsequent fate in the animal
organism, by a series of valuable experiments by A. Schittenhelm.' They
have been confirmed by further observations by Richard Burian.^ We have
already mentioned the fact that Horbaczewski could detect the formation
of uric acid in the pulp of organs, or in extracts of them, in the pres-
ence of oxygen. It was now found possible to follow carefully the

' A. Schittenhelm: Z. physiol. Chein. 42, 251 (1904); 43, 228 (1904); 45, 121, 152,
161 (1905); 46,354 (1905).

2 R. Burian: Z. physiol. Chera. 43, 497 (1905).

THE NUCLEOPROTEIDS.

293

various phases of this conversion. We have also called attention to the
fact that the tissues of many organs contain ferments which are capable
of breaking down the nucleoproteids into their components, and finally
disintegi'ating the separate cleavage-products, such as the nucleic acids,
into their simpler components. The individual purine bases are changed
eventually by ferments into uric acid, as has been unquestionably proved
by the above investigators. Schittenhelm has proved that when the
pulp, or extract of organs, is first boiled, no uric acid can be obtained.
He finally succeeded in isolating the ferment producing uric acid
from the organs. Instead of employing organ extracts, we can now
utilize active ferment solutions for the experiments. The advantage of
this method is evident when we appreciate the fact that the pulp, or
extract of organs, necessarily contains varying amounts of purine bases,
which are practically absent from the isolated ferment or in its solutions.
Schittenhelm obtained xanthine on adding the ferment from beef-spleen
to guanine out of contact with the air, but, by conducting air through the
liquid, uric acid resulted. The transformation of guanine into uric acid
accordingly takes place with the intermediate formation of xanthine. The
following formulae indicate these changes:

HN— CO HN CO HN— CO

NHo

C C— NH —

II II V/

C =0 C— NH

CO C— NH

II II •i^

N— C— N
Guanine

CH

:cH

:co

HN C— N

Xanthine

HN— C— NH

Uric acid

Guanine is converted by hydrolysis into xanthine by the loss of an NHj
group. By the oxidation of xanthine, uric acid is formed. Two ferments,
therefore, participate in the conversion of guanine into uric acid. In the
same manner adenine goes over into hypoxanthine, which is then converted
into xanthine, and the latter into uric acid:

N=C.NH2 HN— CO

HC C— NH ■

II II >«
' N— C— N
Adenine

HN— CO

HC C -NH

II II N

CH

N— C— N
Hypoxanthine
HN— CO

CO C— NH

I II >«
HN— C— N
Xanthine

CO C-NH

\

HN— C— NH

Uric acid

CO

294 LECTURE XIII.

The ferment which converts guanine into xanthine, and adenine into
hvpoxanthine, is widely distributed. It is evidently found in all organs.
The oxidizing ferment, on the other hand, which finally produces the
uric acid, seems to be restricted to individual organs. It has been found
in the spleen, lungs, liver, intestine, muscles, and the kidneys of cattle.
Further investigations have disclosed the remarkable fact that the same
organs of different kinds of animals vary considerably in this respect, so
that a generalization of results obtained with different species of animals
is not permissible. Schittenhelm and Bendix ' showed that the trans-
formation of purine bases into uric acid not only takes place in this way
in glass vessels, but also in the organism itself, by injecting guanine sub-
cutaneously and intravenously into a rabbit. They found a considerable
increase in the uric acid of the urine, and detected the presence there
of a purine base corresponding to xanthine, which is evidently to be
regarded as an intermediate product in the formation of uric acid from
guanine.

Up to this point we have not mentioned an important fact which makes
it difficult to trace the quantitative relations in the production of uric
acid from the purine bases. There are ferments present in many tissues
of the animal organism which are capable of further decomposing the
uric acid formed. Schittenhelm calls them uricoli/tical ferments, in order
to indicate that we are dealing with an entirely different process from
that of the uric acid production. Such a ferment has been found in the
kidneys, liver, and muscles, and very probably also in bone marrow.^
Schittenhelm has succeeded in isolating this ferment. It is evident that
if a destruction of uric acid takes place in the animal tissues, the old-time
conception that the amount of uric acid excreted is an index of the quan-
tity of uric acid formed in the organism, can no longer be accepted. An
increased excretion of uric acid in the urine may, of course, be due to a
greater production thereof; it may, however, also indicate a lessened-
destruction.

The question now arises, What are the degradation products of uric acid
when a decomposition sets in? We can at once answer that we do not
know definitely. Hugo Wiener ' has practically proved that glycocoll
is produced from uric acid when administered to a rabbit. He found
that the reserve supply of glycocoll in this animal was fairly constant.
An increase was noted in the amount of glycocoll excreted after the injec-
tion of uric acid. This increase could be checked by administering ben-
zoic acid, in which case hippuric acid was formed. Wiener has also shown

' Z. physiol. Chem. 43, 365 (1905).

' Cf. also Hugo Wiener, Arch, exper. Path. Pharra. 42, 375 (1899); also Zentr.
Physiol. 18, 690 (1905).

= Arch, exper. Path. Pharm. 40, 313 (1897).

THE NUCLEOPROTEIDS. 295

that the amount of glycocoltin beef kidneys could be appreciably increased
by digesting them with uric acid.* It seems probable that the decom-
position of uric acid may result in the formation of glycocoll; but we,
nevertheless, wish to state that Wiener's investigation is not entirely con-
vincing, for his method of estimating the glycocoll was not an exact one,
and, above all, its production from other sources was not absolutelj'
excluded. As the proteins participate largely in the production of glycocoll,
it is necessarily difficult to estimate the amount of glycocoll originating
from the uric acid.

Urine also contains oxalic acid:

COOH

I
COOH

A part of this undoubtedly originates from the food. Another portion
is unquestionably produced in the tissues. The assumption has often
been made that oxalic acid may be a decomposition product of uric acid,
although no satisfactory proof has been presented to substantiate this
view. From a chemical standpoint such a formation of oxalic acid seems
perfectly possible. We know that uric acid can go over into alloxan,
parabanic acid, oxaluric acid, and finally into oxalic acid. It is impossible
to say anything further about a source of the oxalic acid which does not
come from the food.

AUantoine has often been regarded as a decomposition product in the
hypothetical formation of oxalic acid from uric acid. It was first found
in the allantoic fluid, and later recognized by Wohler ^ as a normal con-
stituent of the urine of suckling calves. Gusserow ' has recently isolated
it from the urine of new-born children. AUantoine is also found in the urine
of various full-grown mammalia; for instance, dogs, cats, and rabbits.

AUantoine is the diuride of glyoxylic acid. It can easily be obtained
by melting glyoxylic acid with urea:

COH /^^H3 /NH^'H-NH^

I -f- 2 CO = CO CO 4- 2 H2O

COOH N^jj^ ^NH— CO NH^^

Glyo.xylic acid Urea .\llantoine

The question of the origin of allantoine has been answered in various
ways. It is generally believed to be related to the purine bodies. This
assumption was strengthened by the discoveries of Salkowski * and Min-

' .\rch. exper. Path. Pharm. 43, .375 (1899).
' .A.nn. 26, 244 (18:58): 70, 229 (1849); 88, 100 (1853).

' .^rch. Gyn. 3, 270 (1872). G. Pouchet: Beitnige zur Kenntnis der Extraktivstoffe
des Urins, Paris, 1880 (pp. 28 and 37).

* E. Salkowski: Zent. Med. Wiss. 36, No. 53, 929 (1898).

296 LECTURE XIII.

kowski,' who showed that when a dog was fed on material rich in purine
bases, such as thymus or pancreas, the amounts of allantoine eliminated
appreciably increased. Cohn ^ found that the administration of hypo-
xanthine to this animal had the same effect. Mendel and White ^ have
proved that intravenous injection of uric acid into cats and dogs, as well
as the injection of salts of nucleic acids, caused an increase in the allan-
toine elimination. Wiechowski * has furnished a better proof that uric
acid is decomposed into allantoine. He succeeded in showing that in
surviving beef-kidney and dog's liver, uric acid is changed quantitatively
into allantoine. This shows one way in which uric acid may be decom-
posed, but it does not necessarily follow that in the decomposition of uric
acid allantoine is formed in every case; perhaps the latter is further
decomposed into urea and glycocoll, and it is quite possible that normally
the decomposition of uric acid may follow an entirely different course.
On the other hand, we can hardly assume that the uric acid is all
decomposed in one way. It is conceivable that, for example, a part is
decomposed with the intermediate formation of glycocoll, while another
part gives rise to allantoine.^

This would give us a clear conception of the formation of uric acid in
mammalia. It can undoubtedly be traced to the degradation of nuclein
substances, and finally to the purine bases. It has never been decided
whether in birds and reptiles (animals in which, as we have seen, the uric
acid in its entire significance and formation takes the place of urea) a part,
if only a very sm.all part, is formed in the way we have just indicated. It
is hardly to be doubted that entirely analogous processes take place in the
organisms of birds and reptiles. Similarly we have no reason for denying
that a synthetic formation of uric acid may also take place in the tissues of
mammals.

Before considering the behavior of the remaining cleavage-products
of the nucleic acids in the animal organism, we must pay some attention
to the purine bases appearing in urine. We have already mentioned,
that, for example, in vigorous muscular activity, such large amounts of
purine bases are transmitted to the blood that the purine content of the
urine is increased. The animal cells do not have time to convert these
bases into uric acid.

Purine bases are constantly present in the urine, some of which are to
be regarded as direct decomposition products of the nucleic acids, while

' O. Minkowski: Zentr. innere Med. 19, No. 19 (189S).

' T. Cohn: Z. physiol. Chem. 25, 507 (189S).

' L. B. Mendel and B. White, Am. J. Physiol. 12, 85 (1905).

* Wilhelm Wiechowski: Hofmeister's Beitr. 9, 295 (1907), 10, 247 (1907.)

' It is interesting to know that allantoine has been found in the bark of tree-
branches and in buds. Cf. E. Schulze and J. Barbieri: Ber. 14, 1602 (1881); J.
pralct. Chem. 25, 145 (1882); Z. physiol. Chem. 11, 420 (1886).

THE NUCLEOPROTEIDS. 297

others are only indirectly related to the decomposition products of nuclei ns.
The amount of such substances present in urine is small and varies. It
may be increased by a diet rich in purine bases. An increased elimination
of purine bases may also result from a greater destruction of leucocytes.
Heteroxanthine,' paraxanthine,' and Z-methylxanthine ^ are purines
which bear no relation to nuclein metabolism. They represent the most
important constituents of the so-called alloxuric bases of urine, and arise
from the caffeine, theobromine, and theophylline present in our table
accessories. The following formulae will give us an idea of the relations
between these substances:

HN— CO CH3 . N— CO CH3 . N— CO

II II II

CO C . N . CH3 CO C . NH CO C . N . CHa

I II )CH I II )CH I II )CH

HN— CN"^ • HN— C.N^ HN— C . N"^

Heteroxanthine ?-Methylxanthine Paraxanthine = 1 : 7-Di-

= 7-Monomethyl- methylxanthine

xanthine

CH3 . N— CO HN CO CH3 . N— CO

I I /CH3 I I CH3 I I

CO c— N :; CO c— N :; co c— nh

I II >H I II )CH I II S(.jj

CHa.N-C-N^ CH3.N C-N^ „„ W N'^

Caffeine Theobromine ^r,;,^ u^-

Theophylhne

The relation of these alloxuric bases to the purine bases of tea, coffee,
and cocoa has been established by means of feeding experiments.

Xanthine, hypoxanthine, guanine, and adenine are among the purine
bases which can be derived directly from the nucleic acids. The two
latter are not invariably present. They evidently appear only when
there is an increased decomposition of material containing nucleins with
relatively less oxidation. Usually they are evidently converted into the
corresponding oxypurines. Xanthine occasionally participates in the
formation of renal calcuH. Pure xanthine calculi rarely occur. Uric
acid is usually a constituent of these bladder stones. These may occur
as small concretionary masses, or as large stones. They are often strati-
fied, in which cases uric acid layers alternate with those of calcium
oxalate.

' G. Salomon: Arch. Anat. Physiol. 1882, 426; 1885, 570. M. Kriiger and G. Sal-
omon: Z. physio!. Chem. 21, 169 (1895); Ber. 16, 195 (1883); 18, 3406 (1885).

^ M. Kruger: Arch. Anat. Physiol. 1894, 553; M. Kriiger and G. Salomon: Z. physiol.
Chem. 24, 364 (1898). ' ,

298 LECTURE XIII.

Two other alloxuric bases, the so-called episarkine ' and epiguanine,^ have
also been isolated from urine. The latter is 7-methylguanine:

NH— €0

NH2 . C C.N. CH3

N C— N^

Epiguanine

CH

We must now attempt to answer the question as to what becomes of the
remaining constituents of the nucleic acids. We are especially interested
in the fate of the three pyrimidines, — uracil, thymine, andcytosine. We
should expect them to be related in some way to the formation of uric acid,
although H. Steudel,' on feeding pyrimidine derivatives to dogs, could not
succeed in transforming them into purine compounds. We know nothing
else that is definite concerning the behavior of the pyrimidine bases in
animal economy.

As regards the phosphoric acid which is obtained by the cleavage of
nucleic acid, we can only surmise what its relations are in metabolism.
Possibly it is utilized in the formation of lecithin.

If we sum up all we know about the breaking down of the nucleoproteids,
and especially of the nucleic acids, we see that there are large gaps in
our knowledge. It is not yet perfectly clear to us how the nucleo-
proteids in the cells themselves participate in metabolism, nor the
function of the nucleus in cell-metabolism. Although the study of the
uric acid formation has given to us ^ fairly clear picture of the transforma-
tion of purine bases, on the other hand it has not been found possible from
this knowledge to shed much light into the obscurity enveloping the
metabolic disturbances which occur in gout and uric acid diathesis. We
can indeed imagine that in these diseases either the production of uric
acid is increased for some reason, or that there is not so much decomposi-
tion of this acid as takes place normally. Now uric acid is very difficultly
soluble in water, and its occurrence in certain tissues — especially in
cartilage — is to be traced to this fact. His* found that one part dissolved
in 39,000 of water at 18° C. We shall study these relations at another
place, and will here merely mention the fact that of the four hydrogen

' Balke: Zur Kenntnis der Xanthinkorper, Inaug. Diss. Leipzig, 1893. Georg Salo-
mon: Z. physiol. Chem. 18, 207 (1894).

2 M. Kriiger: he. cit. Arch. Anat. Physiol. 1894, 553; Z. physiol. Chem. 24, 364 (1898);
26, 389 (1898-99).

= Z. physiol. Chem. 32, 285 (1901).

< Verh. xvii, Kong. Med. p. 315 (1899); Deut. Arch. klin. Med. 67, SI (1900); Verb.
xviii, Kong. Med. 425 (1900) and Zent. Stoffwechsel-und Verdauungskranliheiten, 1,
61" (1900); 3, 434 (1901); His and Paul:.Z. physiol. Chem. 31, 64 (1900).

THE NUCLEOPROTEIDS. 299

atoms which are replaceable by radicals in the uric acid molecule, only
two take part in the formation of salts. Uric acid is, therefore, a dibasic
acid, and forms two series of salts, the acid or monobasic salts, and the
neutral or dibasic salts. Thus we have acid sodium urate, also called
monosodium urate, and neutral or disodium urate. Many speculations
have been brought forward regarding the deposition of uric acid in the
tissues, especially those of the joints, in gout, basing them upon the diffi-
cult solubility of uric acid. On the one hand, an increased formation of
uric acid may account for its elimination, and on the other hand the com-
position of the blood, lymph, and other constituents of the tissues may l)e
such that the uric acid is even more insoluble than under normal conditions.
All of these hypotheses have failed to be very fruitful. They have no
good foundation. For example, we do not know in what form the uric
acid is transported in the blood and tissues. The assumption has been
made that it circulates, not in a free state, but combined with albumin,
nucleic acids, and other substances, although no positive proof has yet
been presented that such is the case. No conclusions can be drawn from
the deposits themselves, which consist of monosodium urate. Great
stress was formerly laid on the increased presence of uric acid in the blood.
We know to-day, that other conditions may result in an increase of uric
acid in the blood without causing the appearance of the symptoms of gout.
Weintraud ' has in fact shown that in a normal individual there is an
increased amount of uric acid in the blood after a diet rich in purine bases.
Again, great stress was laid upon the increased elimination of uric acid in
gout until it was positively shown that it is permissible to speak of an
increased elimination only during an acute attack. Otherwise — aside
from the fact that in gouty diseases the purine values of the urine vary
more than under normal conditions — the amount of purine present in
the urine is practically the same during a long period of time.

It is very noticeable that the deposition of uric acid, especially in gouty
inflammations, seems to be confined to specific locations, such as the
smaller joints of the extremities. The suggestion has been made that
the primary cause of the whole ailment is not a disturbed metabolism of
the purine substances, but an alteration of the tissues at the place in ques-
tion. Just as it has been assumed that the formation of gall-stones is
due primarily to an inflammation of the biliary passages, and likewise that
renal calculi may originate from some organic lesion, the assumption
has also been made that the circulating uric acid was deposited at a given
spot on account of some change in the ti.ssues there.

This is not the place to consider the pathology of gout, nor to enter into
any discussion of the various theories concerning it. We can only attempt

' Wiener klin. Rundschau: 10, Nos. 1 and 2, pp. 3 and 21 (1896). For further liter-
ature see H. Wiener: Ergeb. Physiol. (Asher and Spiro) Jg. 2, 1 Abt., p. 377 (1903).

300 LECTURE XIII.

here to explain pathological processes in the light of physiological-chemical
investigations, and on the other hand obtain from pathological research
new points of view for physiological-chemical work. Doubtless the essen-
tial part of purine metabolism will find its full explanation only by means
of further studies in pathology and in physiology. We must be satis-
fied with merely sketching the existing situation, and bring forward the
fact that for the present there is plenty of room for hypotheses, a sure
sign that the investigations have by no means solved the problem. It
seems certain that gouty diseases will not all be referred to a single cause,
nor to a single disturbance in cellular metabolism. Here, as in diabetes,
various disturbances may take place in different stages of the total purine
metabolism, which will all finally result in the same symptoms.

LECTURE XIV.

THE MUTUAL RELATIONS BETWEEN FATS, CARBO-
HYDRATES, AND ALBUMINS.

I.

In our previous treatment of the three most important classes of organic
food-stuffs, the fats, carbohydrates, and albumins, we have considered each
individual group by itself, as regards the way in which it is absorbed, the
place where it is assimilated, and the relations of the group to specific
functions of the body. We have found that carbohydrates are the most
important source of muscular power, while in the fats we have the princi-
pal source of heat. The significance of the albumins is much less certain.
We know that they are absolutely unreplaceable, and also that they act
as building material for the cells, being necessary to replace the parts
which have been used up. We do not at present understand why the
animal organism requires so much albumin under all conditions, nor
why it so quickly decomposes, up to a certain limit, the ingested
albumin.

By more closely following the metabolism, we quickly encounter results
which do not harmonize, for example, with the assumption that the mus-
cles are only capable of acting by means of the energy given to them by
the carbohydrates. Again, it was noticed early that a portion of the car-
bohydrates disappeared when the diet was rich in these substances, — i.e.,
this part could not be detected in the form of glycogen, — and, moreover,
an exact study of the respiratory exchange showed that no increased com-
bustion of the sugars had taken place. According to this we can only
assume that the e.xtra carbohydrate is retained in some form other than
glycogen.

Before discussing those facts, which force us to the assumption that a
transformation of one group of food-stuffs into another may take place
in the animal organism, we will first of all call attention to the relations
existing between fats, carbohydrates, and albumins, according to our
present knowledge regarding the chemical composition of these substances.
Of course, this will only show us the possibiliiy of such transformations,
and indicate the manner in which they 7nay take place. The organism
itself may, naturally, choose an entirely different course, and utilize other

301 "

302 LECTURE XIV.

compounds, e.g., certain decomposition products, as bridges from one
food-stuff to another.

If we follow the formation of the most varied carbon compounds in
plants, which are the prime source of all carbon combinations in living
organisms, we are forced to the assumption that the first product arising
from the assimilation of carbon dioxide under the influence of chlorophyll
and the sun's energy, is a carbohydrate, or at least some compound very
closely related to this class of substances.' The carbohydrates undoubt-
edly assume a central position in plant metabolism, while the albumins
and fats are of minor importance. But it is not impossible that the
assimilation of carbon dioxide may proceed in various ways, and form
different primary assimilation products. It is indeed possible that the car-
bohydrates, i.e., the large amount of starch present, may conceal other
compounds.

Up to the present time, because starch is detected so easily and so
positively, this and analogous substances' have been of chief interest to
us. On the other hand, the carbohydrates impart to the plant organism
their individuality. We find them in every direction; and when we
consider the numerous polysaccharides (the more simple ones like
starch, which arise from the condensation of a single kind of sugar, and
the other innumerable, more complicated sugars, consisting of unlike
components, such as arabinose, xylose, dextrose, etc.), we will recognize
the fact, immediately, that the carbohydrates occupy the same pre-
dominating position in plants that the albumins do in animal organisms.
At all events, each of the three groups of food-stuffs — fats, carbo-
hydrates, and proteins — • is ultimately derived from the carbonic acid
of the air, for that is the only source worth mentioning of the carbon
in plants.

In considering the assimilation of carbon dioxide by the plants, we
referred to Baeyer's hypothesis, that formaldehyde is to be looked upon
as the first condensation product. We stated then that we could easily
imagine the various different kinds of sugars to result from the condensa-
tion of several molecules of formaldehyde. On the other hand, we also
mentioned Emil Fischer's hypothesis, which includes the possibility that
the glycerose, discovered by him, may occupy the primary stage in the
whole process of carbonic acid assimilation. This assumption has much
in its favor; and if glycerose, perhaps, does not actually appear at this
first stage, we must remember the possibility that it may result from the
disintegration of carbohydrates. At any rate it is interesting to think
that glycerose may perhaps occupy an intermediate position in the further
syntheses of the plant organism. From glycerose on, it is easy to build
bridges to the group of proteins as well as to that of the fats.

' Cf. Lecture IV.

THE MUTUAL RELATIONS. 303

The relations to the fats are evident from the following formulae:

CH2OH COH COOH

i I I

CHOH CHOH CHOH

CH2OH CH2OH CH2OH

Glycerol Glycerose Glyceric acid.

(Glyceraldehyde)

Glycerose is simply the aldehyde of glycerol.' Its preparation from
glycerol was accomplished by Emil Fischer. Conversely, we can imagine
one of the fat components, glycerol, as originating from glycerose. The
relations become much more complicated if we attempt to derive the
fatty acids, the other components of fats, from sugar. Erail Fischer -
suggests their formation in the following manner: To produce oleic and
stearic acids, three molecules of grape-sugar (or six molecules of glycerose)
may unite at their aldehyde groups, thus forming a chain of 3 X 6 = 18
carbon atoms. By rearrangement of the atoms and withdrawal of oxygen,
i.e., by reduction, the above-rnentioned acids may be formed. The other
fatty acids, e.g., palmitic acid, can be formed perhaps in a similar manner,
except that sometimes only hexoses are used for the synthesis, while, at
other times, hexoses and pentoses are both utilized, according to the
number of carbon atoms in the acid molecule. Thus we can imagine
palmitic acid, with its 16 carbon atoms, being produced from one
hexose and two pentose molecules. Pentoses are very widely distributed
in the vegetable world, and in large amounts. They play a far less
important part in the animal body, although they may be formed by
decomposition. We have already learned that a pentose, arabinose, may
be very easily derived from glucose, or from gluconic acid; and on the
other hand, we have seen that an oxidation product of glucose, glucuronic
acid, will easily go over into a five-carbon sugar by splitting off carbon-
dioxide.

Thus, while we are in a position to derive one of the components of the
fats, glycerol, from the carbohydrate group, we have but little foundation
for the formation of fatty acids from the same group. We are at present
confined to hypotheses. We have not yet succeeded by any chemical
means in converting sugars into fat.

' The original glycerose, with which Fischer worked, really contained chiefly
dihydroxyacetone. von Lippraann and others, however, prefer to call glyceral-
dehyde (of which there are two stereo isomers) glycerose rather than the dihydroxy-
acetone.— Tr.\NSL.'VTORS.

' Die Chemie der Kohlehydrate und ihre Bedeutung fur die Physiologie, Berlin,
A. Hirschwald, 1894, p. 28.

304

LECTURE XIV.

Let us consider the relations of glycerose to the proteins, or to their
building materials. The following formulae will be helpful:

COH

I
CHOH

I

CH2OH
Glycerose

CH2OH

I
CHNH2

CH2SH

I

CH . NH2

COOH COOH

a- Amino-/?- Hydroxy- a-Amino-/3-thio-

propionic acid propionic acid
= Serine =Cvsteine

CH3

I
CHNH2

COOH
a-Amino-
propionic acid
= Alanine

These formulae show at once what slight differences exist between these
apparently entirely distinct groups. It would not be at all difficult to
imagine the above three amino acids as being derived from glycerose.
Leucine might be produced from one molecule of a hexose or two molecules
of glycerose, by the addition of ammonia and partial reduction ; ' and by
oxidation, the dibasic glutamic and aspartic acids may be formed from
leucine. We meet with certain difficulties when we attempt to account
for the formation of these last compounds. Leucine, which is isobutyl-
aminoacetic acid, contains a branched chain. The difficulties increase
when we come to proteins containing an aromatic ring, such as phenyl-
alanine and tyrosine. To produce the benzene nucleus from the carbo-
hydrates is a highly complicated process. Such transformations undoubt-
edly do occur in the organism of plants, although the formation of these
substances is, probably, not a direct one.

We must also consider the close relation of alanine to lactic acid, which
is so easily produced by the action of alkalies or ferments.

CH3

CHOH

I
COOH

Lactic acid

CH3

I
CHNH2

I
COOH

Alanine

Plants could also form their albumins or individual amino acids in this
manner. Conversely, lactic acid may be easily formed from alanine,
serine, and cysteine by disintegration; from alanine, for e.xample, simply
by splitting off ammonia. In fact, such processes must take place in the
animal organism to a considerable extent. Lactic acid, therefore, may
arise from two sources in the animal organism. It may come from carbo-
hydrates or from albumin.

The formation of albumin in the animal organism from the other kinds

' Cf. E. Fischer and E. Abderhalden: Z. physiol. Chem. 36, 268 (1902).

THE MUTUAL RELATIONS. 305

of food is of little consequence, or at least all of our present knowledge
of albumin metabolism speaks against it. We need only think of the
possibility of fats and carbohydrates being produced from albumin. Now
we have already seen that a large part of the elementary components of
the proteins are very closely related to the lower members of the normal
fatty acid series. We know further, that only a part of the carbon leaves
the organism in combination with the nitrogen of the individual amino
acids. A portion of the carbon chain must remain behind. What becomes
of this is not yet apparent. Right here the investigation must start con-
cerning the transformations of the albumins into fats and carbohydrates,
for all such changes must result from these carbon chains. We have, to
be sure, no positive proof that such transformations do take place.

Perhaps the intermediate product, glucosamine, may throw some light
upon the formation of albumin from carbohydrates in plants, and, con-
versely, the production of carbohydrates from albumins in the animal
organism. It is a derivative of glucose (or of mannose), and is closely
related on the one hand to the sugars, and on the other, to the oxyamino
acids. It is certainly not without significance, that nature has provided
such connecting links. We recall the following formulae:

CH2 . (OH)

CH, . (OH)

CH2 . (NH2)

1

CH . (OH)

CH . (OH)

1

CH2

1

CH . (OH)

1

CH . (OH)

1

CH2

1

CH . (OH)

1

CH . (OH)

1

CH2

CH . (OH)

1

CH . (NH2)

1

CH . (NH2)

1

CH :0

d-Glucose

CH :0

Glucosamine

COOH
Lysine

CH

I
CH2

I

CH . (NH2)

COOH
Leucine

The carbohydrates, as we have already seen, are related to the poly-
hydric alcohols on the one hand, and to various acids of different char-
acter on the other. We can thus, either directly or indirectly, connect
large groups of different compounds with the sugars. We are still entirely
in the dark regarding the method of formation of innumerable tannins,
ethereal oils, alkaloids, etc., which occur in the plant organisms. All
of these must ultimately be referred to the carbon dioxide of the air.
What relations they possess to the carbohydrates is still entirely beyond
our knowledge; in fact, owing to their great complexity, we are almost
forced to the conclusion that they are not at all related. C. Harries* has

' C. Harries: Ber. 38, 1195 (1905).

306 LECTURE XIV.

shown, however, that products of the vegetable kingdom which appar-
ently are not at all related to the carbohydrates, may, nevertheless,
have been derived from them. He showed that caoutchouc, a compound
containing an eight-carbon ring (1 .5-dimethyl-cyclo-octadi-ine), on
hydrolysis yielded Isevulinaldehyde, CH3 . CO . CH2 . CH2 . COH, and
the ozonide of caoutchouc yielded similarly Itevulic acid. Now, the sugars
readily go over into laevulic acid, while, on the other hand, it is also possible
that caoutchouc may be built up of pentoses. Their reduction to CsHs,
and subsequent condensation into

/CH, . C . CH, . CH., . CH

(11 II )

\ HC . CH, . CH, . C . CH3 /.Y

could account for the formation of caoutchouc. Perhaps the «-methyl-
furan, discovered by Atterberg ' from beech-wood tar, which easily goes
over into Isevulinaldehyde,- may be even more closely related to the caout-
chouc synthesis. At all events, this gives us a new support for the assump-
tion that the whole large group of terpenes may likewise be derived from
the carbohj'drates.

In this connection we must not forget to mention a peculiar compound
which occurs in the vegetable kingdom, as well as in the animal organism,
and has the empirical formula of the hexoses. We refer to inositol (or
inosite). It is found in the muscles, liver, spleen, kidneys, suprarenal
bodies, lungs, brain, leucocytes, and the testes, and has often been noticed
in the urine under normal as well as pathological conditions. Inositol,
CeHioOe, was formerly looked upon as a sugar. Maquenne ^ later recog-
nized it as a derivative of hexamethylene, with the following composition:

CHOH— CHOH

CHOH^ V'HOH

^ CHOH— CHOH ^

Hexahydroxybenzene = Inositol

It is indeed possible that we have in this case, one of the first stages in
the conversion of a carbohydrate into the benzene ring, so that this
compound which is of so much biological interest opens up further possi-
bilities for the formation of the numerous aromatic compounds in the
plant organism. Although chemical investigation has often served to
give us better understanding concerning many obscure biological processes,
we must not forget that a clear conception is lacking in regard to the most

■ A. Atterberg: ibid. 13, 879 (1880).

' C. Harries: ibid. 31, 37 (1898).

» Maquenne: Compt. rend. 104, 1719 (1887); ibid. 109, 812 (1889).

THE MUTUAL RELATIONS. 307

important changes. We are obliged to resort here almost exclusively to
experiments with animals. Certain observations are also a result of
Nature's physiological experiment, pathology. In all such investigations,
we are dealing with indirect methods of proof. Experiments with animals
rarely lead to other than indirect results. These are not entirely con-
clusive; they are merely more or less convincing, and in most cases are
dependent upon the personal interpretation of the investigator. This is
a weak point in nearly all biological investigation, which undeniably gives
it a certain amount of fundamental uncertainty. For this reason, it is
difficult to form a positive decision from the numerous experiments which
have been performed in the attempt to decide the question as to the
conversion of one class of food-stuffs into another. We can merely
enumerate here the more important and best substantiated conceptions
and those experiments which have been carried out in the most convinc-
ing manner. On the other hand, we would be committing a grave error
if we were to consider only those processes and changes in the organism
as proved for which we have a purely chemical explanation, and which we
are able to repeat, where possible, outside of the living cell. We would
thus be making a restriction, which would hinder the further develop-
ment of biology, and we should then be forgetting that biology has a
distinct field of its own, with its own peculiar methods of investigation.
Ultimately, of course, we must always depend upon the exact sciences,
chemistry and phj-sics, and consider a biological problem as fully settled
when these sciences supply the key-stone.

Let us begin first of all with the carbohydrates and their conversion into
the other two groups of food materials, fats and albumins. The formation
of albumin from carbohydrates in vegetalile organisms comes into con-
sideration onh- to the extent that the plant cells utilize the carbon chains of
the sugars in syntheses together with the nitrogen, which is either assimi-
lated by the roots from the ground, or, as in rare cases, is obtained directly
from the air. We do not know anything definite about these processes.
In the animal organism, we can deny the po.ssibility of such transforma-
tions. It is otherwise, however, as regards relations of the carbohydrates
to the fats. One of the first observations in this connection was the
formation of fat in ripening rape-seeds, and in the pulp of the olive. We
know that unripe rape-seeds contain large amounts of carbohydrates, but
practically no fat. During the ripening of the seeds we find that the
carbohydrates gradually diminish, while fat takes their place. This obser-
vation is very significant, for the seed is unable to give off its carbohydrate
externally, or to take up fats. The changes in nature of a food material are
also attested b\' the metabolism. The respiratory quotient changes. Ger-
ber' has shown that the ratio of carbon dioxide produced to the oxygen

' C. Gerber: Compt. rend. 125, 658 and 732 (1897).

308

LECTURE XIV.

consumed, in the unripe seeds, is less than one. More oxygen is taken up
than is given off as carl^on dioxide. During ripening the ratio becomes
greater than one, only to fall below one again after the fruit has become
completely ripe. Analogous results have been ob.served in the ripening of
olives, which in their unripe condition contain mannitol. The following
figures published by A. Roussille ' will give us an idea of the process which
accompanies the ripening of olives:

June 20 . . .
July 30 . . .
August 30
September 30
October 30 .
November 25

Per cent.

Per cent.

1.40

5.49

29 19

14.619

62 30

4.189

67.21

4.411

68.57

4.329

Leclerc du Sablon ^ found the following amounts of carbohydrates and
fats in nuts per 100 parts of the dry substance:

Water.

Oil.

Glucose.

Saccharose.

• Amylose.

July 6

837.

3.

7.6

0.

21.8

August 1 . . . .

535.

16.

2.4

0.5

14.5

August 15 ... .

274.

42.

0.

0.6

3.2

September 1 . .

48.

59.

0.

0.8

2.6

October 4 . . . .

10.

62.

0.

1.6

2.6

The changes of carbohydrates into fat in the almonds were as follows
on a basis of 100 parts of dry substance:

Date.

Water.

Oil.

Glucose.

Saccharose.

Amylose.

June 9

July 4

August 1 . . . .
September 1
October 4 . . . .

896.
716.
219.
117.
12.

lo:

37.
44.
46.

6 0

4.2

0.

0.

0.

6.7
4.9
2.8
2.6
2.5

21.6
14.1
6.2
5.4
5.3

It is interesting to note that fatty acids — evidently as intermediate
products — have been observed during the formation of the fats.' How
the transformation, as a whole, is effected, has never been explained.

We also know that the reverse process, i.e., the formation of sugars from
the fats, also takes place, and this has been followed in the case of germi-

' A. Roussille: Md. 86, 610 (1878).

' Ihid. 123, 1084 (1896).

3 von Rechenberg: Ber. 14, 2216 (1881).

THE MUTUAL RELATIONS. 309

nating rape-seeds. If we permit these to develop away from the light, we
observe the fat disappearing from the cotyledons. In its place we find
carbohydrates: starch, cellulose, gum, sugar. If we allow seeds, rich in
starch, to germinate in glass tubes sealed under mercury, no change in the
volume of gas can be observed. During the germination of seeds contain-
ing oil, on the other hand, we notice that gas is being consumed, from
the fact that the mercury rises. This corresponds to the amount of
oxygen which is required to convert the fats into carbohydrates which are
richer in oxygen. These important observations were made by Julius
Sachs and Wiesner ' in 1859.

There is, therefore, no doubt that the plant cell is capable of pro-
ducing carbohydrate from fat, and, conversely, fat from carbohydrate. If
we assume with Kassowitz ' that the protoplasm absorbs, or assimilates,
one of these compounds, e.g., the fat, in order subsequently to form
in this case, the carbohydrate, in such a way that there is no direct con-
nection between the two compounds, then we miss the point at issue, and
the whole process is beyond our understanding, for it cannot be a matter
of indifference for the functions of the protoplasm, whether at one time
fat, at another time carbohydrate, and yet again albumin, is at its disposal.
The question concerning the transformations of the separate food-stuffs
is only postponed by such assumptions, and it becomes more involved and
obscure.

A much disputed question is this: Does the animal cell possess the same
abilities as the plant cells? Can the animal organism convert fats into
carbohydrates, and, conversely, carbohydrates into fats? The last ques-
tion has been aaswered in the affirmative. We know that with a diet
rich in carbohydrates, an appreciable part of the ingested carbohydrate
is not stored up in the form of glycogen, although there is no glucohemia.
The fact forces us to the conclusion that carbohydrates can be stored away
as reserve material in some other form than glycogen. Numerous feeding
experiments have shown that an accumulation of fat follows a diet com-
posed largely of carbohydrates.' This decision may be reached in two
different ways: first, by determining the amounts of fat formed with
a diet containing a definite quantity of fat, albumin, and carbohydrate;
and secondly, by estimating the daily elimination of carbon dioxide. The
first proof has been carried out, as a rule, in the following manner: Two

' J. Sachs: Bot. Zeit. 1859.

' Cf. M. Kassowitz: Allgemeine Biologie (3 vols).

' Cf. also B. Schulze: Landw. Jb. 1, .57 (1882'). F. Soxhlet: Z. Landw. Vers. Bayern.
August (1881). St. Chaniewski: Z. Biol. 20, 179 (18.84). H. Weiske and E. Wild:
ibid. 10, 1 (1874). I. Munk: Arch. Path. Anat. 101, 91 (1886). E. Meissl and F.
Strohmer: Sitzber. Akad. Wiss. Berlin, 88, HI (July. 188.3), and Monatsh. 4, 801 (1883).
E. Meissl, F. Strohmer, and N. v. Lorenz: Z. Biol. 22, 63 (1886). C. Voit: Sitzungsber.
Miinchener Akad. 1885, p. 288. M. Rubner: Z. Biol. 22, 272 (1886).

310 LECTURE XIV.

dogs from the same litter and about the same weight, are permitted to fast
for quite a length of time. One of the animals is then killed, and the
amounts of albumin and fat contained in the carcass are determined.
The other animal, which is assumed to possess approximately like quan-
tities of the above substances, is then fed for a time on a definite diet,
the composition of which in fat, albumin, and carbohydrate is known.
The unabsorbed part of this material can be estimated by an analysis of
the fajces. After several days, this animal, which has now gained in weight,
is killed, and the amount of its fat and albumin determined. In this
manner it can be shown that as much as 85 per cent of the ingested starch
has been converted into fat. ■ We must not place too much confidence in
these values, for the assumption that the one animal, at the termination
of the fasting period, contained exactly the same amounts of fat and
albumin as the other, is open to question. It is remarkable, nevertheless,
that there should be such great differences between the organisms of the
two animals, and that corresponding results have been obtained in a
number of experiments.

Thus, Tscherwinsky ' obtained the following values:

Albumin.

Fat.

Animal fed 4 months with barley contained

2.52 kg.
0.96

9.25 kg.
0.69

First animal, therefore, gained

The food contained

1 . 56 kg.
7.49

8.56 kg.
0.66

-5.93 kg.

+ 7.90 kg.

The animal used in the experiment, a young pig, had, therefore, gained
7 . 9 kilograms fat, which could not have come from the fat in the food,
and certainly not from the albumin. Carbohydrates, therefore, were trans-
formed into fat.

We can, as we have said, also follow the respiratory exchange in an
animal fed on a diet rich in carbohydrates. If we know the amounts of
carbon, albumin, and carbohydrate present in a food poor in fat, but rich
in carbohydrates, we can, on the one hand, by determining the nitrogen in
the urine, estimate the amount of albumin retained in the body, and, on
the other hand, we can estimate the quantity of carbon remaining in the
organism, by determining the amount exhaled as carbon dioxide in addi-
tion to that eliminated as urea. In this way it has been found that the
amount of carbon retained may be so large that the only possible explana-
tion is that the ingested carbohydrates have been converted into fat.

The production of fat from the sugars has, a priori, much in its favor.

' N. Tscherwinsky. Landw. Vers-sta. 29, 317 (1883).

THE xMUTUAL RELATIONS. 311

The animal organism has only a certain amount of room in its tissues for
the carbohydrates. The quantity of glycogen which can be stored up in
the liver, muscles, and the other organs, is very limited. The animal
organism is often provided with larger amounts of carbohydrate than it
is able, at the moment, to utilize. It is here that the depots for fat storage
are utilized. Large amounts of carbohj^drates after having been trans-
formed into fat can be held in reserve until needed. We have not the
slightest idea where, in the animal organism, or in what organ, this trans-
formation takes place. It is possible that the liver executes this com-
plicated process.

Although it is generally admitted that the animal cell is able to convert
carbohydrates into fats, the reverse process is a much disputed problem.
We are accustomed to look upon most chemical reactions as reversible.
We also know that the animal cells are capable, directly or indirectly, of
performing characteristic processes. They build up and they tear down
material only to reconstruct it, and, finally, by its complete destruction,
they utilize the energy contained in the food. We have seen representa-
tives of all three classes of nutrient materials break down in the intestine,
and, again, we have traced their reconstruction into more complicated
compounds. The cells of the liver produce glycogen from grape-sugar,
permitting it to become glucose again as it is required. Another problem
is this: Is the animal organism, under normal circumstances, capable of
satisfying its carbohydrate requirements from fats. This would hardly
be the case under ordinary conditions, for, a priori, as we shall later on see
more in detail, there is no reason at hand why the fat should first go
over into carbohydrate, in order that the organism may utilize its energy
for specific purposes. On the other hand, the possibility of such a trans-
formation unquestionably exists. We can imagine that each cell can only
utilize certain compounds for specific functions. Thus, we may assume
that the muscle cells operate only by means of carbohydrates. The selec-
tive action of the ferments would support such an assumption. We know
that, in manj' cases, they are unable to act upon substances closely related
to compounds that they can decompose. Every ferment seems peculiarly
fitted to attack only certain definite compounds. Thus it would be easy
to understand that the muscle cells, which are especially adapted to act
upon carbohydrates, can utilize only such potential energy as is presented
to them in this form. On the other hand, we must remember that the
cleavage and combustion of the food materials are not the source of the
liberated energies, but only act as a loosening momentum, or as a shock.
The true cause is the chemical energy of the food substances — ultimately
the sun's energy, transformed sunlight. We cannot understand, a friori,
why the energy liberated by the combustion of the fats could not be
just as well utilized by the muscle cells as that which ari.ses from the

312 LECTURE XIV.

carbohydrates. If the muscle cell possesses the ability of consuming
the fats, it hardly seems probable that it must first convert the fats
into carbohydrates in order to abstract the energy from them.

If the fats were first changed into carbohydrates before they could be
utilized for work, then, according to Zuntz, a diet of fats exclusively would
require about 30 per cent more energy to perform a given amount of work
than would be required after a diet of carbohydrates. This, in fact, is not
the case.'

Until recently the above discussion would have iseen quite unnecessary.
The general conception was that the different food materials in the organ-
ism, i.e., in the tissues, and finally in the cells, were burned up directly
by the aid of oxygen. It was only necessary that the food material and
the oxygen should both be present in such form that they could react
together.

The whole problem thus became a very simple one. The conviction is,
however, becoming more and more pronounced, that the relations are
much more complicated. The cells themselves participate far more
actively in the combustion of the food materials than we have hitherto
imagined. They prepare the substances for disintegration and combus-
tion. The fact that our foods are not attacked l:)y the oxygen of the air,
has led to the assumption that the o.xygen is " activated " in some manner
in the organism, and that this activated oxygen effects the combustion.
This problem of the activation of oxygen has been the main topic of
interest, whereas the behavior of the food materials themselves, in the
oxidation process, has been entirely neglected. Now it has only recently
been shown, as we have seen, that the diabetic possesses, as far as our
present knowledge is concerned, an absolutely normal capacity for oxida-
tion. Grape-sugar alone he consumes more or less imperfectly. As soon,
however, as any slight attack has been made upon the glucose molecule, —
as soon as it becomes "opened up," — the diabetic is able to consume com-
pletely, and to utilize, the energy contained therein.^ We must consider
the possibility of a change occurring in the food by means of a cell influence
— either directly or indirectly by means of ferments — before the oxidation
takes place by the oxygen, which is carried to the tissues with the blood.
The material is first of all " opened -up " so that it can be oxidized. How
this takes place, we do not yet know. We can imagine, however, that
the oxygen is first attached to the " opened " molecule, which is then
destroyed. We know, from the investigations of C. Harries,' that caout-
chouc, under the action of ozone, goes over into a compound rich in

' H. N. Heineraann: Pfluger's Arch. 83, 44 (1901). J. Frentzel and F. Reach:
ibid. 83, 477 (1901).

' Cf. Lecture V, p. 101.

' C. Harries: Ber. 37, 2708 (1904); ibid. 38, 1195 (1905).

THE MUTUAL RELATIONS. 313

oxygen, the so-called " ozonide," which can be looked upon as a peroxide

of laevulinaldehyde.

0 : C (CH3) . CHo . CHo . CH : O

II II

0=^- O

It decomposes into Ifevulinaldehyde, liEvulic acid, and hydrogen peroxide,
on prolonged boiling with water. It is clear that, in the animal organism,
such an energetic action of oxygen can hardly take place on the unaltered
food materials, although it is possible that the cell might so change the
food as to make it more susceptible to attack. Such a conception corre-
sponds more nearly to the actual functions of the cells. It consumes the
substances which it needs from time to time; it must, therefore, have a
direct influence on these processes. Under normal circumstances, oxygen
is invariably present. The cell, i.e., its protoplasm, either activates the
oxygen, or else it seizes the separate food materials as it needs them, and
offers them to the oxygen for oxidation. At any rate, the decomposi-
tions, which have been observed directly, e.g., that of the carbohydrates,
may be traced back directly to cell-activity. The cell is, therefore, able
in two ways to regulate exactly its requirements of energy: by the activa-
tion of the oxygen, or by the preliminary preparation of the food for
combustion.

We have intentionally gone into this matter somewhat in detail, in order
to show that the combustion of the food materials in the tissues is not
necessarily such a simple process as we have heretofore assumed. For
these reasons it is, as yet, impossible to decide whether fats are normally
converted into carbohydrates. Such a conversion is hardly probable. It
is more natural to imagine a direct utilization of the energy in fats by the
animal cell, and especially by the muscle cells.

Experiments have been made with animals in the attempt to settle the
question whether fats are converted into carbohydrates. Before discussing
these we must show in general what deductions can be safely drawn from
such investigations.

Investigations on the formation of sugars from other compounds than
carbohydrates, and especially from fats, have, as a rule, been carried out
in one of two ways. We can make the animal chosen for experiment free
from glycogen, by fasting, hard work, or strychnine convulsions, and then
feed it the compound which is to be tested as a glycogen-former. By
suksequently determining the glycogen content of the whole animal, it is
possible to find out whether any glycogen has been formed. Only in
exceptional cases, however, have these experimental conditions been satis-
fied. Rarely was it satisfactorily proved that the animal was free from
glycogen at the real beginning of the experiment, and such a source of
error is serious on account of the small amount of glycogen which is found

314 LECTURE XIV.

at the end of the test. Again, the methods employed in determining the
amount of glycogen were inaccurate in many cases. Finally, the experi-
menters have been satisfied, for the most part, with merely estimating
the amount of glycogen in the liver, entirely neglecting that which might
be retained in the other organs. This is on the assumption that the liver
is the place where the most glj^cogen is formed. We do not know, how-
ever, how the animal organism behaves when it has been deprived of its
carbohydrate stores. It is perfectly possible that the sugar will first go
to those organs which need it the most. The chisf objection to all these
experiments is always that the ingested compound may have had an
indirect effect, i.e., when we add fat, to determine whether fat can produce
glycogen, and we do, as a matter of fact, find that this causes an increase
in glycogen, then the objection can be raised that perhaps the fat, which is
itself consumed, has acted as an albumin-sparer, so that the glycogen
may have been produced from albumin. The method of proof is, in every
case, an indirect one, and this makes it far .more difficult to arrive at the
correct conclusion. In one and the same experiment, the formation of
sugar may be traced back to either the fats or albumins, according to the
point of view. The same may be said of the second method of carrying
out the experiment. In this case, glucosuria is first produced, and then
the influence of various substances on the elimination of sugar is studied.
Thanks are due to E. Pfliiger' for critically examining all of the investi-
gations which have been made up to the present time that have any
bearing on this subject, thereby showing with great clearness that the
whole problem is still in a ver}^ uncertain state.

First of all, glycerol was tested with reference to its ability to produce
sugar; i.e., in other words, the problem was to decide whether the animal
cell is capable of synthesizing sugar from glycerol. This compound, as we
have already seen repeatedly, is related to sugar in its composition. We
have mentioned the hypothesis that glycerose is the starting-point in the
formation of glycerol by the plant cell, and in the same way we can regard
glycerose as resulting from glycerol when the plants convert fat into
sugar. Now the old idea that only the plant cells are capable of effecting
synthesis, has long since been set aside. We know that the animal organ-
ism is also able to build up. There is, therefore, no reason why sugar
could not be formed from glycerol. Of the many experiments which have
been performed in this direction, we will refer to those of Cremer ^ and
LUthje.^ They fed dogs, whose pancreas had been removed, with glycerol,
and determined the increased elimination of sugar in the urine. Liithje

' E. F. W. Pfliiger: Das Glycogen ii. s. Beziehungen z. Zuckerkrankheit, 2d ed. Bonn.,
M. Hager, (1905). Cf. E. Pfliiger: Pfliiger's Arch. 103, 1 (1904).

' M. Cremer: Sitzber. Gesel. Morph. u. Physiol. Miinchen, May 27, 1902.
' H. Luthje: Deut. Arch. klin. Med. 79, 498 (1904); 80, 101 (1905).

THE MUTUAL RELATIONS. 315

administered as much as 350 grams per day. The animal under e.xperi-
ment, a dog, weighed 15 kilograms. If, in accordance with common
experience, we assume that this animal had stored up 11 grams of glycogen
per kilogram of weight, at the beginning of the experiment, we obtain, as
the total amount of glycogen present, 165 grams, corresponding to 183
grams sugar. If we take the maximum value, 40 grams of glycogen
per kilogram weight, we have 800 grams glycogen, corresponding to 664
grams sugar.' The animal under experiment, however, excreted 1408.4
grams sugar. With the first assumption, 1225 grams, and with the
second, 744 grams, of sugar thus remain unaccounted for. The ingested
albumin and glycerol must be regarded as producing this sugar. The
animal excreted 209.8 grams nitrogen during the whole period of the
experiment. Liithje calculated that, at mo,st, 630 grams of sugar could
have been produced from the conversion of the albumin. This leaves
considerable sugar still unaccounted for unless we admit that it came from
the glycerol. Liithje's experiment is the only one which really proves
that glycerol can be converted into sugar.^ All of the other investigations,
namely, those which showed an increase in the glycogen content of the
liver, are not above criticism. The discovery that the animal cell is
capable of transforming glycerol into sugar is another connecting link
between the animal and plant cells. We must not, however, deceive
ourselves with the thought that the conversion of glycerol into sugar takes
place in normal metabolism to any considerable extent. We know it is
true that fats, before being absorbed, are more or less disintegrated into
their components, glycerol and fatty acids. We also know that neutral
fats are formed again in the intestine. A small amount of free fatty acids
remains, and likewise some glycerol. There is but very little, however.
It might be possible for fat to undergo combustion without previous
hydrolysis, and the glycerol therein converted into glucose instead of being
consumed with the rest of the fat molecule. The amount of such glycerol
would be only 1 1 per cent of the neutral fat, and this is relatively little.

The next question that arises is whether the fat itself, i.e , the fatty
acid component also, can go over into sugar. Direct experiment indicates
that this is not the case. Fat itself does not cause anj^ increase of sugar
in the urine, even in the most severe cases of diabetes. E. Pfliiger, who
has recently come to the conclusion that a source of sugar other than the
carbohydrates themselves must be looked for in bad cases of glucosuria
and diabetes, nevertheless holds to the opinion that fat can produce sugar.
He explains the fact that fat does not cause any increased excretion of

' E. Pfliiger calculates as a maximum 61.5 <rrams sugar. CSee CtlycoKen, he. cit. p. 5/57.')
' More recently Karl Griibe, Pluger's Arch. 118, 1 (1907), has shown that glycerol

after having been passed through the liver of a turtle caused an increase in the

glycogen content of this organ.

316 LECTURE XIV.

sugar during diabetes, by the behavior of fat materials in the general
metabolism. In the first place, it is brought forward that the animal
organism does not regulate its consumption of energy according to the
quantity of food administered; that is, the combustion in the organism can-
not be increased beyond certain limits by the amount eaten. The amount
of energy utilized is dependent on the work that the organs have to do.
This regulates the amount of material consumed. If more food is eaten
than is utilized, the excess can be stored up. If all food is withheld from
an animal, it will live largely upon its store of fat. A.n appreciable fall-
ing off in metabolism immediately follows, which cannot be entirely
recovered simply by a diet of fat. This may, however, be accomplished
if a little albumin is added. Voit has also shown that fat metabolism
ceases when a sufficient amount of albumin is fed to an animal. It
will then live solely at the expense of albumin. This observation applies,
strictly speaking, only to the carnivora. The omnivora and herbivora are
unable to handle as much albumin as the metabolism requires. They con-
sume carbohydrate and fat in proportion as the albumin alone is insuffi-
cient to fulfill all of the requirements. The quantities of carbohydrate and
fat changed over are regulated by the supply of albumin at hand. " The
extent of albumin metabolism is dependent on the amount of albumin
presented; while fat metabolism is independent of the fat supply."' Fat
changes are regulated chiefly by the supply of albumin, and, secondarily,
the amount of carbohydrates. Every excess of fat is deposited. The
reason that a diet of fat does not cause any increased elimination of sugar
in a dialjetic patient, is due to the fact that the fat consumed cannot go
beyond prescribed limits. By feeding fats we only produce an accumula-
tion thereof. The formation of sugar from fat is regulated exclusively
by the amount of work performed by the cells which oxidize fat to sugar.
In order to trace the influence of fat upon the elimination of sugar, there
remains the method proposed by E. Pfluger.^ A dog, with extirpated
pancreas, is fed exclusively on albumin. If the eliminated sugar is derived
from the body fat, we would expect, as soon as the animal had been freed
of its fat, that the elimination of sugar would cease. Pfluger's experi-
ments in this direction led to no definite conclusions, for his animals all
died at a time when the fat supplies were about exhausted.

Now what is the origin of the sugar which the animals in the experi-
ments of Pfliiger and others always eliminated, even when all carbo-
hydrates were withheld from them? Liithje, to whom much credit is due
for his extensive investigations regarding the production of sugar during
diabetes, is of the opinion that the albumins in the food are the source of
the sugar in such experiments. This lirings us to the relations of the albu-

E. Pfliiger: Glycogen, he. n't. p. .329.

E. Pfliiger: Pfluger's Arch. 108, 115 (1905).

THE MUTUAL RELATIONS.

317

mins to the carbohydrates. Clinical experience has long since decided
that albumin will produce sugar during diabetes. In no case, however,
is the proof clear and free from criticism. We are compelled to state
that at present we know nothing definite concerning the way sugar is
formed from albumin. It has been attempted to get around this problem
by placing especial stress upon the carbohydrate groups present in the
proteins. We have, however, already called attention to the fact that
the amount of carbohydrate groups present in proteins is of little quanti-
tative significance. To be sure, the carbohydrate content of the different
albumins from a practical standpoint will vary greatly, for w-e do not
feed animals with " pure " albumins, but albumin in the form of meat,
etc. Even then, however, if we were to a.ssume that these albuminous
substances may contain as much as 10 per cent of carbohydrates, this
would not explain the appearance of the large amounts of sugar which are
eliminated by diabetics. E. Pfliiger himself has brought forward the best
proof in this direction, by feeding dogs with codfish, which is practically
free from glycogen and sugar.' This flesh contains 0.55 per cent fat.
The following table gives a summary of such an experiment:

Daily Elimi-

nation of

Daily

No. of

Average

Daily In-

Nitrogen in

.Wer-

D'

Period of Time,

Days

Weight of

take of

Grams

age of

Nitrogen

1905.

in Each
Period.

the Dog
in Grams.

Nitrogen
in Grams.

Sugar

Balance.

N

in the

in the

Grams.

Urine.

F»ces.

1. Jan. 14-17 . .

4

8580

15.2

13.2

4.2

27.8

- 2.2

2.10

2. Jan. 18-30 . .

13

8350

27.9

24.6

2.5

55.9

-f- 0.8

2.27

3. Jan. 31-Feb. 15

16

8300

36.1

30.8

4.1

69.8

-1- 1.2

2.26

4. Feb. 16-Feb. 20

5

8200

36.8

31.1

6.3

71.2

- 0.6

2.29

5. Feb. 21-Feb. 26

6

8150

39.1

31.0

6.3

47.3

-1- 1.8

1.52

6. Feb. 27-Mar. 4

6

7070

8.8

16.2

4.5

35.5

-11.9

2.20

Here the case is perfectly clear. The question to decide is: Does the
eliminated sugar arise from fat or from albumin; i.e., to be more precise,
from the amino acids in the albumin? Only these two classes of food-stuffs
can be taken into consideration, and not the insignificant amount of carbo-
hydrate groups. The reverse process, the production of amino acids
from carbohydrates, undoubtedly takes place in plants. We have already
become acquainted with the close relations existing between glycerose (as
well as lactic acid, a derivative of the carbohydrates) and alanine, serine,
and cysteine, and have learned incidentally that the relations betw-een the
other known albumin decomposition products to the carbohvdrates still
remain unknown. The plant cells could hardly be considered as produc-
ing carbohydrates from albumin. Conversely, the animal cell unques-

' E. Pfluger: Pfliiger'.s .\rch. lOS, p. i:36. ' t'f. page 321.

318 LECTURE XIV.

tionably does not synthesize albumin from carbohydrates. It does not
follow that the reverse process may not take place. We must also remem-
ber that the proteins contain large complexes, the nature of which we know
but little. It is possible that more complicated hydroxy-acids, such as di-
amino-tri-hydroxy-dodecoic acid,* are present, the conversion of which
into carbohydrates would be easy to understand. At all events, we must
admit that the conversion of amino acids into sugars is no more difficult to
understand than is the transformation of fatty acids into carbohydrates,
and conversely. The formation of sugar from amino acids is connected
with the question as to what becomes of the carbon of the amino acids
which does not leave the organism in the form of urea. When the func-
tion of these nitrogen-free carbon chains is explained, the problem of the
sugar formation of sugar from albumjn will be less difficult to solve. This
is the vital point of the whole question, and from this standpoint the whole
subject must be considered.

We might think that feeding amino acids alone would show, in the first
place, whether they have any effect upon the formation of glycogen; and
secondly, whether they effect the elimination of sugar. Such experiments
have, in fact, been made. Alanine and leucine, on account of their having
respectively three and six carbon atoms in the molecule, seemed especially
suited for the experiment, although the latter has a methyl side chain.
It is well known that normal carbon chains of the carbohydrates can easily
go over into branching chains, — for example, in the formation of saccharine;
and so, on the other -liand, we can imagine the possibility of the reverse
process taking place in the transformation of leucine into a sugar. The
experiments which have been performed in this direction are very con-
tradictory, and have led to no positive conclusions,^ although apparently
the feeding of individual amino acids, in particular leucine and alanine,
does not cause any accumulation of glycogen.

This does not by any means preclude the possibility of sugars being
produced from amino acids. We can easily imagine that the disintegration
of the amino acids from a protein proceeds in its intermediate stages in a
different manner from that which takes place when we feed large amounts
of the individual amino acids, as such, to the organism. We know very
little as yet about the intermediate disintegration of albumin, and are
unable to state whether the amino acids, as such, are set free, or whether
the disintegration of the proteins does not take place after the removal

' E. Fischer and E. Abderhalden: Z. physiol. Chem. 42, 540 (1904).

» Cf. E. Pfluger's criticism (Glycogen, loc. cit.). R. Cohn, Z. physiol. Chem. 28, 211
(1899), found that feeding rabbits with leucine caused an increase of over 400 per cent of
glycogen, whereas O. Simon, ibid. 35, 315 (1902), found no glycogen formation to take
place. Grube (Pfluger's Arch. 118,1 (UX)7) ) also came to the conclusion, from experi-
ments with the livers of turtles, that leucine and alanine did not increase the glycogen
content.

THE MUTUAL RELATIONS. 319

of the amino groups and oxidation. We should undoubtedly be making a
great mistake if we were to draw the conclusion that all such compounds
are decomposed in the same way, merely because in mammals the greater
part of the nitrogen from ingested protein, amino acids, and polypeptides
appears in the urine. We must not forget that the formation of urea
represents only one phase in the disintegration of the amino acids. It
certainly does not explain the intermediate albumin metabolism.

The formation of sugar from amino acids has been regarded as a safe
conclusion on account of the fact that these acids are closely related to
the fatty acids, and for this. reason it may seem superfluous to make such a
sharp distinction between the formation of sugar from fats and from albu-
min. On the other hand, the objection may be raised that the amino acids
which have been studied are derivatives of the lower fatty acids. If we
assume that the amino groups are removed from the amino acids while they
are in the tissues, thus forming fatty acids, it would be expected that an
accumulation of glycogen would result on feeding animals with the fatty
acids in question. L. Schwarz ' has carried out such experiments, and
found that the fatty acids administered did cause an increase in the
acetone bodies eliminated, but not in the sugars.

Embden and Salomon - have recently investigated the influence of
individual amino acids (alanine, glycocoll) of asparagine and of lactic
acid on the elimination of sugar in dogs whose pancreas had been removed.
They found that an increase resulted. It is possible that the sugar was
formed, in this case, from the above compounds, and that this kind of
experiment is more suitable for establishing the formation of sugar than
the method of studying the glycogen formation. It is certain that sugar
will be formed from albumin or its amino acids only in proportion as sugar
is needed by the organism. Further experiments will be nece.ssary to deter-
mine whether the administered amino acids have acted in a direct or
indirect manner.

The experiments with the amino acids themselves, therefore, are not
as yet such as lead to definite conclusions. We are thus brought back to
our former question: Is sugar produced from albumin itself? Claude
Bernard made experiments on the formation of glycogen after feeding
albumin. He showed that a dog, which had been fed for months only on
meat, contained large amounts of glycogen in its liver. He also raised
fly-maggots upon boiled egg-albumin or extracted meat, and found large
amounts of glycogen.' E. Kulz * repeated these last experiments. He
divided 72 fresh eggs of Musca vomitoria into two equal groups, and

■ L. Schwarz: Arch. Win. Med. 76 (1903).

' G. Embden and H. Salomon: Hofmeister's Beitr. 6, 63 (1904); ihid. 6, 507 (1904).

' C. Bernard: Lec^ons sur le Diabete, p. 464 (1877).

' E. Kulz: Pfluger's Arch. 24, 71 (1881).

820 LECTURE XIV.

immediately determined tlie amount of glycogen in one of these, while
the remainder were cultivated upon the albumin from hens' eggs. He
could not observe any formation of glycogen in this case, although positive
results were obtained when the maggots were nourished with meat. These
experiments do not furnish a direct proof that sugar is formed from albu-
min. We know now that neither the albumin from hens' eggs nor from
meat is free from sugar. It is possible that the glycogen produced by the
maggots results from the sugar in the food. Numerous feeding experi-
ments with animals fed exclusively on albumin in the form of meat, etc.,
have, without exception, led to the conclusion that glycogen is produced
from albumin.' It would take too long to dwell upon all of these experi-
ments. Many of them can be thrown out because there was sufficient car-
bohydrate present in the food to account for the glycogen found. On
the other hand, in many cases it was not shown that the animals under-
going experiment contained no glycogen at the beginning of the test.
Finally, we must add that improvements in the methods of estimating
glycogen have shown the unreliability of values formerly obtained. Thus,
8.5 grams of glycogen was assumed as an average content per kilogram
weight of a dog. Pfltiger increased this value to 11 grams, while to-day
we regard 41 grams as more nearly correct.

It will be sufficient if we compare the two series of experiments of
E. Pfliiger and H. Liithje,^ which are free from criticism. We have already
given Pfliiger's figures, and will add merely that he in one experiment,
for example, found the following balance:

Total sugar formed 3097 . 1 grams

Explainable as residual glycogen 422 . 3 grams

Sugar formed from other sources 2674.8 grams

This result corresponds with that of Liithje, and proves positively that
sugar must have been formed from some other source than carbohydrates.
Liithje also fed casein to a dog whose pancreas had been removed. The
animal weighed 5.8 kilograms. It eliminated 1176.7 grams of sugar
between October 2 and November 24. E. Pfliiger ^ subtracts 650.6 grams
from this as being due to sugar present in the food and from the glycogen
originally present in the body. It must be said that this value subtracted
by Pfliiger is, if anything, too high rather than too low.^ Thus, at least,

' Cf. E. Pfiuger: Glycogen, loc. cil. p. 240, etc.

2 H. Liithje: loc. cit. Deut. Arch. klin. Med. 79, 4999 (1904). Pfliiger's Arch. 106,
160 (1904).

' E. Pfliiger: Pfliiger's Arch. 106, 168 (1904).

* E. Pfliiger calculates 109 grams sugar from 328 grams of serum-albumin, basing
this on the values obtained from mucin by F. Miiller. Serum-albumin and serum-
globulin contain, at the most, 2 per cent sugar. If we take out 7 grams sugar as due
to the serum-albumin, we would undoubtedly be placing the sugar value too high
rather than too low. Liithje administered 4100 cubic centimeters serum. Since 1000

THE MUTUAL RELATIONS. 321

526 grams of sugar remain unaccounted for. LUthje assumes that this
means that albumin forms sugar. This assumption of Luthje corresponds
to the fact that the elimination of nitrogen, and likewise that of sugar,
increases uniformly when the administration of albumin is increased.
The ratio of the sugar (dextrose) eliminated to that of the nitrogen in the

urine is usually designated by the symbol — ■

This ratio is assumed to have a constant value, namely, 2 . 8.' E. Pfluger
calls attention to the fact that the value is not as constant as is generally
supposed, but that very appreciable deviations may arise. Thus, in some
of his experiments, Pfluger found the value to be less than 1, and in
other cases it rose as high as 14.6. Furthermore, it must be remembered
that every increase in the amount of albumin administered results in the
saving of a corresponding amount of fat and carbohydrate. Every addi-
tion of albumin lessens the extent to which carbohydrates are oxidized.
Now diabetics, as well as animals afflicted with glucosuria, have not lost all
of their ability to consume sugar. Some work is performed in both cases
at the expense of energy present in sugar. If now albumin be fed in large
amounts, this can be burned up in place of the carbohydrates formerly
required. The result of this will be that more unconsumed sugar circu-
lates in the blood, and is, therefore, eliminated. This also would account
for the parallel elimination of sugar and of nitrogen.^

If we consider all that we know with regard to the formation of sugar
from substances other than carbohydrates, we arrive at the conclusion that
it is at present impossible to decide whether fats or proteins must be drawn
upon as a further source of supply. We know merely that sugar can be
produced from one of these two classes of compounds. From a chemical
standpoint, the transformation of fatty acids into sugar is just as com-
plicated as is that from the amino acids; yes, we may say, that the con-
version of the oxyamino acids into carbohydrates is easier to understand,
because glucosamine may be regarded as a compound intermediate betv/een
the two latter groups. On the other hand", we must not forget that a very
large part of the albumin molecule is composed of simple amino acids.

The question as to whether sugar is produced from fats or from albumins
is probably an unnecessary one. We can see no particular reason why
both should not be utilized in this way, according to the conditions. Such
an assumption would explain the observed irregularities, and enable us to

understand why the quotient — should at one time be less than 1, and

grams serum would not have over 1.5 grams sugar in a free form, 4100 cubic centi-
meters must contain less than 6. 15 grams.

' O. Minkowski; Arch. exp. Path. Pharm. 31, S5 and 97 (1892).

' E. Pfluger: Glycogen, loc. cil. p. 325. Cf. also Lecture I, pp. 6 and 7.

322 LECTURE XIV.

again more than 2.8. There is apparently no reason why we should
say that either the fats or albumins do not produce sugar; and when we
find one author regards fats, and another albumin, as the sole producer
of sugar other than carbohydrates, it only means that there is no direct
proof of a formation of sugar from either one of these substances, but
there is merely an indirect proof of their influence. Too much depends
upon the interpretation of the results. Whether sugar is formed normally
from fat or albumin has not been proved by any of the experiments. It
is possible that the organism of the diabetic and that of the dog suffering
from glucosuria, may behave in entirely different ways.

We must consider the possibility that the glucosuria produced by
different causes may have different effects. We do not know the organs
in which the transformation of albumin or fat into sugar takes place.
This does not preclude the possibility that the process is carried on for
both substances in the same place, nor that the transformation of one
material is more affected than another in any given case of glucosuria.
An observation of G. Rosenfeld ' may have a bearing on this subject. He
caused a dog weighing between 3 and 5 kilograms to fast for five days,
and then on the sixth and seventh days injected 2 or 3 grams of phloridzin.
Carbohydrates were fed to the dog at the same time. The dog was killed
on the eighth day. The liver showed no indication of fat infiltration. If,
on the other hand, there is no carbohydrate in the food, but fat, or nothing
at aU, is fed, a decidedly fatty liver is obtained. While the amount of fat
in the liver of a fasting dog is about 10 per cent of the dry substance, the
livers of animals used for the last experiment have shown from 25 to 75
per cent of fat. The glycogen had shrunk to small proportions. The
fatty liver disappeared two days after the injection of the phloridzin.
In these cases, as was shown by microscopic examination, the fat
was not deposited in the connective tissues, but directly in the liver
cells. Here it is a transference of fat from other organs of the body, as
was shown by Rosenfeld, and not a transformation of glucose or albumin
into fat.

It is possible that this phenomenon has some connection with the con-
version of fat into sugar, although it need not apply to glucosuria due to
other causes. No fatty liver results, for example, in glucosuria caused by
extirpation of the pancreas. It, therefore, appears, a priori, wrong, to place
the various forms of glucosuria and diabetes upon the same basis, simply
from the fact that they have in common the predominating symptom of
glucohemia and the resulting glucosuria. The widely different causes of
the different kinds of glucohemia cannot be sufficiently emphasized. It is
possible that a study of an individual case in different directions, rather

' G. Rosenfeld: Verb. Kong, innere Med. 359 (1893).

THE MUTUAL RELATIONS. 323

than following solely the elimination of sugar, would more likely lead to
a solution of the problem.'

In this connection, the question arises whether all carbohydrates are
able to form glycogen. We have already seen that glucose and fruc-
tose are glycogen-formers. We also know that milk-sugar and cane-
sugar,^ on being introduced into the blood, are eliminated unchanged in
the urine. Cane-sugar is normally disintegrated in the intestine. The
usefulness of milk-sugar is evidently dependent on the presence of lactase,
as E. Weinland ' has shown. Glycogen does not seem to be formed from
pentoses.* There is a large amount of uncertainty concerning these ex-
periments on account of the fact that a compound which causes an accu-
mulation of glycogen, need not necessarily itself participate in its pro-
duction. The combustion of the substance may indirectly shield, for
example, glucose from oxidation, thus causing deposition of glycogen.
Although this objection may seem uncalled for, there is much in its
favor. Perhaps only those sugars are glycogen-formers which are capable
of going over into glucose; for grape-sugar is, probably, the only building
material of glycogen. All of those compounds which can be changed
into it, must be looked upon as producers of glycogen.

We now reach the problem as to the relations of albumins to fats. Is
albumin converted into fat? There is, a priori, no reason why such a
change should be impossible. We know that the nitrogen in urea only
carries with it a part of the carbon present in the albumin, while the larger
part of the carbon in albumin is transformed in the body in some other
way. It is conceivable that the-se other carbon chains may be deposited
in the form of fat under certain conditions, in which case a direct fat
accumulation might result from a diet of albumin. The assumption of
the formation of fats from albumin would be of considerable value with
reference to the production of sugar from albumin or fat, for those inter-
mediate compounds which lead to the synthesis of fat from albumin may
also be cjosely related to the formation of sugar. We should, as a rule, be
rather cautious in assuming such complicated changes, especially in view
of the rapid progress of albumin metabolism. On the other hand, the old
ban, which was so long placed on the animal cell as not being in any man-
ner capable of effecting a synthesis, will gradually have to be withdrawn.
It is desirable, therefore, to establish more proof from other points of view.

' One might expect the problem to be solved by following the respiratory exchange.
Unfortunately there are no convincing investigations at hand. Cf. E. Pfhiger: Pflijger's
Arch. 108, 473 (1905); Magnus Levy: Z. klin. Med. 56, 83 (1905).

2 F. Voit: Deut. Arch. klin. Med. 58, 523 (1897).

= E. Weinland: Z. Biol, 38, 16 and 606 (1899); 40, 386 (1900). Cf. also R. II. A.
Plimmer: J. Physiol. 34, 93 (1906).

* E. Salkowski: Z. med. Wiss. 11 (1893). Z. physiol. Chem. 32, 393 (1901).

324 LECTURE XIV.

The conversion of albumin into fat was for a long time looked upon as
an established fact, albumin being even considered as the main source of
the body fat. This conception originated with Voit and Pettenkofer,'
as an outcome of their experiments on metabolism. Since the time when
E. Pfliiger^ critically examined the values given by the above authors,
the belief in the production of fat from albumin has been more and more
doubted, this being especially true since many observations, undertaken
in order to show such effects, have indicated that the above conclusions
were erroneous. Pettenkofer and Voit fed dogs with meat as free as
possible from fat. They found all of the nitrogen of the ingested albumin
present in the excretions, but only a part of the carbon. It was natural
to conclude from this, as we have already indicated, that the portion of
carbon which did not leave the organism in combination with the nitro-
gen, was utilized for the production of fat. Pettenkofer and Voit reached
this conclusion owing to the fact that they had assumed 1 : 3.68 to be the
relation of nitrogen to carbon in meat free from fat. Pfliiger, after allow-
ing for glycogen, reduces this value to 3.22, while Rubner places it at 3.28.
If we apply these changed values to the results of Pettenkofer and Voit,
we find that the assumption that fat is produced from albumin no longer
has any support.

M. Kumagawa' has recently tried to solve the problem in the following
manner. He caused two dogs from the same litter to fast for 24 days.
One of the animals was then killed and analyzed. The second dog for
quite a long period was fed a liberal supply of horse meat (49 kilograms
in 49 days). The body weight rose from 6.08 to 10 kilograms. The
fat content of this animal at the beginning of the experiment must
have been about 120 grams. The other dog contained this amount.
The animal under experiment showed 1087 . 7 grams of fat on being killed.
The meat fed to this dog, however, contained 356 grams of glycogen and
1084 grams of fat. The amount of fat in the food, therefore, would of
itself have been sufficient to cause this increase.

.\lthough we must admit that no satisfactory proof has been obtained
by experiments on metabolism as regards the transformation of albumin
into fat, we must not forget, on the other hand, that these results merely
give us a rough idea of the whole interchange of material, but never the
finer details of cell activity. It is still a very remarkable fact that such a
small portion of the carbon in albumin should leave the organism in com-

' M. Pettenkofer and C. Voit: Ann. Supp. 57, 361 (1862). C. Voit: Z. Biol. 5, 106
(1869); 6, 371 (1870); 7, 433 (1871). Handbuch der Physiologie des Gesamt-
stoffwechsels und der Fortpflanzung, Leipsic, 1881. Ueber die Ursachen der Fett-
ablagerung im Tierkorper, Munich, 1883.

' E. Pfliiger: Pfluger's Arch. 50,98 (.3.30 and 396) (1891); 51,229 (1892); 52, 1
(1892); 68, 176 (1897); 77, 521 (1899).

' M. Kumagawa: Communication from the University of Tokio (1890).

THE MUTUAL RELATIONS. 325

bination with the relatively large amount of nitrogen. This must certainly
possess some deep significance. Let us consider leucine:

^CH . CH2 . CH . (NH,)COOH,
CHs^
and urea:

/NH2

C=0

The nitrogen united to C = O in the latter compound, is derived from
two molecules of monoamine acid. What becomes of all of the rest of
the carbon chain? We know nothing about this nitrogen-free residue of
the amino acids. It is possible that it is immediately consumed. This
has never been satisfactorily proved. On the other hand, we can under-
stand the fact that nitrogen leaves the organism in the form of urea, from
the standpoint that the organism has utilized the energy of the albumin
to the fullest possible extent. An elimination of carbon chains containing
nitrogen would indicate an incomplete combustion of available material.
The fact that the disintegration of the albumin in this manner is a very
economical one, — a loss of energy always occurs in the urea itself, — does not
permit us to form any opinion regarding the utilization of the remainder
of the carbon. We call attention to these relations, because the whole
question of the relation of albumins to fats and carbohydrates is dependent
upon this, and we wish to emphasize the point that the assumption of the
albumin being rapidly consumed in toto, is not sufficiently supported by
the facts.

Let us consider those investigations and observations which are held
to prove that fat may be produced from albumin. We must state, at the
start, that a large number of such investigations are worthless, owing to
the fact that the methods employed for estimating the fat do not give
any idea as to the actual fat content of any individual organ. The fat
in the organs is evidently present in different forms. A part of this may
easily be extracted by ether. If we examine a piece of tissue which has
been freed of fat in this manner, we obtain the impression that all of the
fat has been removed. This is, however, not the case; for if we digest the
residue with pepsin-hydrochloric acid, or boil it with 2 per cent hydro-
chloric acid, we can extract some more fat with ether. Instead of using
ether for extraction purposes, it has been recommended to employ alcohol
and chloroform alternately.' The fact that the fat is not entirely extracted

' C. Dormeyer: Pfliiger's Arch. 65,90 (1897). J. Nerking: ibid. 73, 172 (1898);
85,330(1901). O.Frank: Z. Biol. 36, 549 (1897). E. Voit: *i<i. 36, 555 (1898).
E. Bogdanow: Pfluger's .\rch. 86, 389 (1897). G. Rosenfeld: Z. in. Med. 33 (1900).

326 LECTURE XIV.

by the ether, is partly because some of the fat is inclosed in the cells,
■while another portion is undoubtedly present in combination with other
substances. In many of the investigations dealing with this problem,
this latter fact has not been sufficiently taken into consideration in the
estimation of the total fat present.

The old observation of the formation of grave-wax, or adipocere, has
been brought forward as evidence of the production of fats from albumin.'
By this process we understand the production of a wax-like mass from a
corpse. The albumin disappears little by little frorn the muscles, and fat
takes its place. This characteristic change is especially noticeable in moist
burying-places, where a slow decomposition proceeds in the presence of a
minimum supply of oxygen. By carefully following this process, and
especially by direct experiments, it has been proved that there is no such
conversion of albumin into fat, but that the fat already present in the
body is responsible for the production of adipocere. This is mainly due
to the fat present already in a given locality, and to such infiltrated fatty
masses as may be deposited there by the water. Moreover, if it had been
proved that the fat arose from albumin in this process, it would possess
no bearing on the production of fat from albumin in the living animal
organism. It would be conceivable that lower organisms, such as bacteria,
etc., are capable of effecting such conversions. The formation of fat from
albumin during the ripening of cheese, for example, is attributed to the
interaction of fungi.-

Hoffmann,^ however, has carried out a notable investigation, which is
considered a proof of the transformation of albumin into fat. He collected
the eggs of flies, and determined the amount of fat present in a number of
them, while the others were cultivated upon defebrinated blood. This
contained 0 . 032 per cent of fat, and the fly-eggs 4 . 9 per cent. The maggots
cultivated upon the blood finally showed at- least 10 times as much fat as
was present in the eggs and blood. Two objections can be raised, for, in
the first place, the method used for estimating the fat is not entirely
aljove criticism, while, in the second place, this conversion of albumin into
fat may have been brought about by means of bacteria, which developed
rapidly in the blood. 0. Frank,^ who repeated the experiment, using meat.

' Cf. J. Kratter: Z. Biol. 16, 455 (1880). Erraan: Vierteljahresschrift gericht
Med. 37, 51 (1882). E. Salkowski: Festschrift fiir Virchow's Jubilaura, p. 23 (1891).
K. B. Lehmann: Sitzber. physikal. med. Ciesel. Wiirzburg (1888). E. Voit: Sitzber.
Gesel. Morph. und Physiol. Miinchen, 4, 50 (1888). F. Kraus: Arch. e.xp. Path.
Pharmak. 22, 174 (1887).

' K. Windisch: .\rbeiten aus dem Kaiserl. Gesundheitsamte, 17 (1900). H.
Jacobsthal: Pfluger's Arch. 54, .584 (1893).

' F. Hofmann: Z. Biol. 8, 153 (1872).

' O. Frank: loc. cit. Z. Biol. 36, 549 (1897).

THE MUTUAL RELATIONS. 327

which had been freed as much as possible from fat, could not obtain any
definite results. The fat formed may very easily have been derived from
the fat of the meat. These experiments also are not suitable to establish
the fact that fat is produced from albumin.

Finally, it may be mentioned that the fat contents of the secretions,
those of the lacteal and sebaceous glands, have repeatedly been referred
to albumin as their source. Direct experiments do not confirm this
assumption. It is also difficult to decide this question, owing to the fact
that the animal organism has stores of fat at its disposal, and is not depend-
ent on the fat in the food. Moreover, the conversion of carbohydrates
into fat must be taken into consideration.

Now we happen to know of a number of processes in which organs,
containing, as far as the eye can see, hardly any fat at all, are sudtleniy
permeated with it. This is especially noticeable in the case of the liver.
We have already met with an infiltration of fat in this organ. We have
seen, that, .as the glycogen disappears after glucosuria has been brought
about by phloridzin, fat will take its place, provided, of course, that no
carbohydrates are present in the food. We have here a physiological
infiltration of fat. It is especially noteworthy owing to the fact that it
disappears again on discontinuing the poisoning b}' phloridzin. Normal
liver cells, capable of performing their functions, will remain.

We know of a large number of poisons, such as phosphorus, arsenic,
antimony, chloroform, alcohol, English oil of pennyroyal, etc., all of
which are capable of causing a local accumulation of fat. Finally, pathol-
ogists are well acquainted with the appearance of a " fatty degeneration,"
which follows various disease processes (influence of to.xins, etc.). We can
very easily imagine that the appearance of acute accumulations of fat in
places where we would hardly expect to find any, would lead to the con-
clusion that some substance had been changed into fat at that place. As
the body cells are generally composed of albumin, it seemed to prove that a
transformation of alliumin into fat had taken place. This conception re-
mained for a long time undisputed, until it was shown both macroscopically
and microscopically, that this fat could have no connection with the true
fat content of an organ. A ti.ssue apparently free from fat may yet contain
as much as 20 per cent, and an organ seemingly laden with fat may
actually possess less than an apparently fat-free tissue. It was necessary
first to trace the formation of fat by exact quantitative methods. A
great advance was also made in the discovery that it was not sufficient
to determine the fat content of any specific organ, but that the amount
of fat in the entire animal must be taken into consideration. We can pass
over the earlier experiments in this direction, which were characterized
by questionable methods for estimating the fat, and confine ourselves to
the more recent work.

328 LECTURE XIV.

Athanasiu ' determined the fat content of 124 frogs. He then poisoned
a like number of animals with phosphorus, and again estimated the amount
of fat in the entire collection. He found no increase in the total quantity
of fat. Taylor,^ in fact, observed an actual decrease.

Experiments with mice gave the same results/ whereas control animals,
which were fed in the same manner, showed from 13.8 to 29.3 per cent;
those which had been poisoned with phosphorus gave only 4.13 to 7.9 per
cent of fat. The organism had, therefore, been deprived of fat. The
livers of those mice which had been poisoned with phosphorus showed
from 7.4 to 37.4 per cent, while this organ in normal mice gave only 5.1
to 11.8 per cent of fat. All of the other tissues had lost fat while the liver
had gained. From this it is easy to assume that the increased fat content
of the liver must stand in direct relation to the diminution in the fat supply
of the remaining tissues. Rosenfeld * has proved this to be so by direct
experiment. He observed fat, from a deposit in a dog which had been fed
with mutton tallow, to migrate directly into the liver, and the composition
of this fat was that of the fat deposited in the dog's own tissues. He also
showed that fasting dogs and hens after being poisoned with phosphorus
gave no indication of any increase of body fat. Their fat deposits had
already been drained. Thus, tlie old idea that albumin is converted into
fat after poisoning with phosphorus must be discarded.

We have already called attention to the fact that the cleavage-products
of albumin, such as tyrosine, leucine, glycocoll, etc., appear in the urine
after phosphorus poisoning. This circumstance seems to support the old
idea that albumin goes over into fat. In fact, a superficial observation
could easily lead to that conclusion. Albumin is decomposed, while fat
appears in its place. The decomposition of the albumin, however, may
take place entirely independently of the fat infiltration. It is clear, on the
one hand, that the great derangenient in metabolism caused by phos-
phorus poisoning also affects the disintegration of the albumins, and
causes destruction of albumin; while the observed amounts of amino
acids, on the other hand, have no relation to the amount of fat present.
Athanasiu could not observe any increased disintegration of albumin,
although a noticeable infiltration of fat had taken place in the liver. The
fattening of other organs, the muscles, heart, etc., can be explained
in the same manner, while the infiltrations of fat due to other harm-
ful influences in no case point to a formation of fat from albumin. In

' J. Athanasiu. Pfliiger's Arch. 74, 511 (1899).

2 Taylor: J, exp. Med. 4, .399 (1899).

' F. Kraus and A. Sommer: Hofmeister's Beitr. 2, 86 (1902).

* G. Rosenfeld: Verhandl. Kong. in. Med. (1894). Allgem. med. Zent. No. 60
(1897); No. 89 (1900). Cf. Fettbildung, Part II. Ergebnisse Physiol. (Asher and
Spiro), Bergmann, Wiesbaden 2 (190.3), p. 50 et seq.

THE MUTUAL RELATIONS. 329

every case there is enough fat ah-eady present to explain these fat
accumulations.

Considering all the results of physiological and pathological experiments
with regard to the formation of fat from albumin, we must admit that
up to the present time no proof has been found which compels us to
assume a transformation of albumin into fat. We must not neglect to
remark that, although the fat present in the body is sufficient to explain
the accumulations of fat, still, this fact does not preclude the possibility
of its production from some other substance. We arrive at the above
indirect conclusion as it seems most probable to us. We would, however,
be making a grave error were we to consider the problem of the produc-
tion of fat from albumin as finally solved. Our comments only go to
show that the experiments and methods so far utilized are insufficient
to confirm such transformation. New questions and new points of view
are necessary for this problem.

We have now reached the end of our observations concerning the con-
version of one substance into another. In the animal organism we are
absolutely certain only of the production of fat from the carbohydrates.
The reverse process, as well as that of the production of sugar from albu-
min, has not yet been sufficiently demonstrated to warrant any definite
decision. At any rate, it will be necessary to look for some other source
than the carbohydrates for an explanation of the elimination of sugar
during severe cases of glucohemia. We have developed the assumption
that both fat and albumin must be taken into consideration, and that the
great difTerences of causes of glucohemia may correspond to different
origins of sugar. Finally, we have seen that there is no absolute proof
that the formation of fat is at all related to the presence of albumin.

We have intentionally avoided giving any definite opinion on these
important questions, preferring to take the opposite stand corresponding
to uncertainty of the facts in hand. Nothing can hurt the progress of
knowledge more than the desire to reach conclusions on such complicated
questions from purely theoretical considerations. We are greatly indebted
to E. Pfltiger, whose critical observations have been followed in every
case where it seemed as if the question had been positively answered. He
has considered all the previous experiments, and has to a certain extent
repeated them.

The relations are evidently entirely different in the plant. It must
manufacture its carbohydrate, fat and albumin, all from the same raw
materials. It easily converts carbohydrates into fats, and fats into
carbohydrates. It also undoubtedly synthesizes its albumin from sugar
and its derivatives. The future can alone decide whether there is any
marked fundamental difference between the activities of the plant and
animal cells, or whether any difference between them may be only quan-

330 LECTURE XIV.

titative rather than qualitative. At any rate, the fact that the animal
cells convert carbohydrates into fat indicates that they possess the same
functions as do the plant cells. The transformation, whether it be from
fat or from albumin into carbohydrate, is a very complicated process, and
it is safe to assume that the animal cell is far more efficient than we have
hitherto imagined. We shall have to take such relations more into
account and synthetic processes will receive more attention in the future.
It is only by reason of far-reaching changes, through disintegrations and
reconstruction, that we are able to understand why every species of animal,
in fact, every individual, should possess a specific composition in spite of
the fact that each may have had the same food.' This alone is sufficient
to warrant us in assuming complicated synthetic processes as taking place
in the animal cell.

Cf. Lecture XXIX.

LECTURE XV.

MUTUAL RELATION BETWEEN FATS, CARBOHYDRATES,
AND ALBUMINS.

II.

The Law of Isodynamics.

So far we have considered only the most important organic nutrients
from one point of view, namely, their chemical composition, and sought
out those facts which led to the assumption that one class of substances
goes over into another in the animal organism. In such cases extensive
chemical changes take place — reduction, oxidation, analysis, and synthe-
sis — before one substance can replace another. This, however, is not
the only way in which one substance may appear in place of another.
The substitution may be a purely physical one; i.e., the energy imparted
to the body by the substances in question may be the most important
factor. In other words, we can conceive that the different organs, e.g.,
the muscles, do not work with one single food material alone in perform-
ing their prescribed functions, but with representatives of all three groups
of nutrients. We could indeed imagine, as we have already mentioned,'
that every individual cell in the body is so adjusted that it works only with
a definite material. We would then have to assume that the substitution
of one of these combustible substances by another must be preceded by a
transformation into the former. When we recall the facility with which
the vegetable organism converts carbohydrates into fats, and fats or
albumin, or both together into sugars, such a conception would, a priori,
not seem so improbable. On the other hand, in such a case the organism
evidently would not work economically, if it were called upon first of all
to produce deep-seated and far-reaching transformations, before it could
utilize the food materials. The entire metabolism would thus resolve
itself into an extremely complicated process, and such transformations
would make themselves felt especially in the case of a restricted diet with
a definite kind of food, e.g., of fats, which are deficient in oxygen, or of
carbohydrates, which are rich in oxygen. One might cite the diabetic as
proof of the fact that an organ can perform its work with the most varied

Cf. Lecture XIV, p. 311.

331

332 LECTURE XV.

kinds of food, for the diabetic can perform muscular work, even though
he is not able, in proportion to the severity of the disease, to utilize to
advantage the most important source of energy, the carbohydrates. For
him the carbohydrates scarcely come into consideration as a source of
energy. Evidently the diabetic performs his work at the expense of other
food material. We cannot look upon this proof as entirely convincing,
for it is precisely those observations dealing with diabetics which have led
to the assumption, that sugar is produced from the two other organic
nutrients: albumin and fat. Why does the dia!>etic patient produce
sugar from these? Why does he not utilize these according to their
calorific value? Why does he carry out the extremely complicated
chemical changes, some of which are so difficult to understand? Cer-
tainly not in order to eliminate more sugar. These transformations must
have a deeper significance. Is this a normal process which comes to light
because of an insufficient utilization of the products formed, or are we to
look upon the production of sugar from fat and albumin as a derangement
of the entire metabolism? These are enigmas, the solution of which still
reaches into the far-distant future. At all events, we repeat once again,
nowhere else does the question appear as vital as here, as to whether
specific atomic groupings are not normally utilized for definite purposes.
Nothing is of greater promise in the entire field of biological investigation
than the clearing up of just such uncertainties and contradictions. Such
work must lead to new problems and new results. We have, on the one
hand, the claim that large amounts of sugar arise from fats and proteins,
■while, on the other hand, there are numerous exact statements which make
it seem improbable that such conversions are, as a rule, necessary in normal
metabolism for the accomplishment of definite work.

If we, for the moment, leave out of consideration the significance of the
organic nutrients as building material for the worn-out or new cells, we
find that their most important function is that of a source of energy.
Chemical energy is transmitted to the animal organism by means of
the food. Mechanical work is performed by the transformation of this
energy. Only a part of the energy is utilized in this way. Another part,
and in fact a very considerable one, is changed into heat. The animal
cell can utilize these sources of energy in two ways: first, by cleavage,
and second, by oxidation. Only a portion of the energy can be trans-
formed into kinetic energy by the former method; oxidation alone
furnishing the possibility for a complete utilization of the energy. Now
the different organic food-stuffs (carbohydrates, fats, and albumins) possess
different amounts of energy; i.e., they have different fuel values. The
amount of energy possessed by the various food materials can be deter-
mined by the quantity of heat which they liberate when undergoing
combustion. This is generally expressed in calories, a small calorie (cal.)

FATS, CARBOHYDRATES, AND ALBUMINS. 333

being that amount of heat required to raise 1 gram of water from 0° to
1° C. A large calorie (Cal.) is the amount of heat necessary to raise 1 kilo-
gram of water from 0° to 1°C. We shall use the large calorie in every
case to express the heat values of individual food materials.

Complete oxidation of one gram of each of the following articles of food
in a calorimetric bomb gave the following values:

Calories

Ca.sein 5.86

Egg-albumin 5.74

Conglutin 5.48

Average value for protein 5.71

Animal ti.ssue-fat 9.50

Butter-fat 9.23

Cane-sugar 3.96

Milk-sugar 3.95

Glucose 3.74

Starch 4.19

The values given for the carbohydrates and fats represent exactly the
amount of heat which is liberated during combustion in the animal organ-
ism. The animal cells likewise oxidize the carbohydrates and fats to
carbon dioxide and water. The physiological heat value of the fats is
generally given as 9.3 calories, and of the carbohydrates 4.1 calories
for each gram of substance. The values in the table do not apply to the
albumins as oxidized in the living organism. The animal cell does not
utilize completely the energy present in albumin. A portion of this energy
goes to waste, usually in the form of urea. We are indebted to Rubner '
for an exact estimation of the physiological heat value of albumin. He
fed a dog exclusively on washed meat, whose heat value had been care-
fully determined. From this he subtracted the heat values of the urine
and faces, as well as that necessary for the swelling of the alliuminous
material and for dissolving the urea. In like manner Rubner determined
the heat of combustion of the decomposed albumin in the body of a fasting
rabbit. He found the following values for the physiological heat of com-
bustion for each gram albumin:

One gram of dry substance — Calories

Albumin from meat 4.4

Lean meat 4.0

Albumin during fasting 3.8

The physiological heat of combustion is not identical for the different
proteins. The normal value for animal albumin is estimated as 4.23

' Max Rubner: Z. Biol. 21, 250 and 337 (1885). Berthelot and Vielle: Compt. rend.
102, 12S4 (1886). Berthelot and Recoura: ibid. 104, 875, 1571 (1887); Berthelot and
Andre: llO, 884 and 925 (1890). F, Stohmann: Z. Biol. 31, 364 (1895).

334 LECTURE XV.

calories; 3.99 for vegetable albumin; and 4.1 as an average value for
proteins as a class.

Before discussing the significance of these figures, we must consider
whether the law of the conservation of energ}^ ' applies entirely to the
animal organism. We have seen that plants, with the assistance of the
kinetic energy of the sun's rays, are able to liberate oxygen from water
and carbon dioxide. They use up kinetic energy and form potential
energy.^ The reverse process takes place in the animal organism. In it the
oxygen unites with the compounds poor in oxygen, the end products being
water and carbon dioxide again. This applies, at least as indicated above,
to the fats and carbohydrates. Potential energy is utilized and kinetic
energy takes its place. This appears partly in the form of heat, partly
as mechanical work. We may expect that the sum of the energies of the
metabolized food materials will be exactly equivalent to the energy pro-
duced by the animal organism.

The first experiment in this direction was carried out by Lavoisier,' as
early as 1780, with, to be sure, rather primitive methods. Neither he
nor the two later investigators Despretz * and Dulong ^ were able to
establish a satisfactory agreement between the amounts of energy received
and that produced. We owe to Max Rubner ° the first exact proof of
this relation, while more recently W. 0. Atwater ' has repeated the exper-
iments, eUminating all sources of error. Atwater compared the amounts
of potential energy in the substances which were actually oxidized in the
body with the amount of kinetic energy evolved by the latter. This
appears in the form of heat in the rest experiments, and as heat and mus-
cular work in the work experiments. Even in the latter case this was
measured in heat units. The following table gives a summary of some of
Atwater's results, the experimental details of which we shall discuss in
another place.*

' Cf. R. Mayer: Die Mechanik der Warme. Stuttgart 1867 (2d ed. 1874); Die
Erhaltung der Energie. Berlin, 1889.

^ Tliis, of course, only applies to the principal activity of the parts of the plants
containing chlorophyll. These, also, require oxygen and give off carbon dioxide (Cf.
Lecture IV).

5 Lavoisier et de la Place: Mem. Acad. roy. sciences, p. 355 (1780).

* Despretz: Ann. chim. phys. 27, 337 (1824).
' Dulong: ibid. (3) 1, 440 (1841).

• Max Rubner: Z. Biol. 30, 73 (1894).

' W. Atwater: Ergeh. Physiol. (Asher and Spiro) Jg. Ill, 1 Abt. p. 497 (1904).
» See Lecture XXVII.

FATS, CARBOHYDRATES, AND ALBUMINS.

335

COMPARISON OF INCOME AND OUTGO OF ENERGY IN 45 EXPERIMENTS

ON METABOLISM, COVERING 143 DAYS. AVERAGE

AMOUNTS PER DAY.

Subject and Kind of Experiment.

Number ol
Experi-
mental
Days.

Net In-
come (Po

tential
Energy of
Material
Oxidized
in Body).

Net Outgo
(Kinetic
Energy
_ Given
off from
Body).

Diflerence (in Terms
of Net Income).

Calories.

Calories.

Per cent.

Ordinary Diet.
Rest experiments:

7 experiments with E. 0. . .
1 experiment with A. \V. S. .
3 experiments with J. F. S. .
1 experiment with J. C. \V. .

25
3
9
4

2268
2304
2118
2357

2259
2279
2136
2397

- 9
-25

+ 18
+ 40

-0.4
-1.1

+ 0.8
+ 1.7

Average for experiments . .

Work experiments:

2 experiments with E. 0. . .
4 experiments with J. F. S. .
14 experiments with J. C. W. .

41

8

12
46

2246

3865
3539
5120

2246

3829
3540
5120

0

-36

+ 1
0

0

-0.9
0
0

Average for 20 experiments .

Average for all rest and work
experiments with ordinary
diet

66

107

17
6
3

4682

3748

2313
2308
2124

4676

3745

2319
2356
2123

- 6

- 3

+ 6
+ 48

- 1

-.01
-0.1

Special Diet.
Rest experiments:

6 experiments with E. 0. . .
3 experiments with A. W. S. .
1 experiment with J. F. S. . .

+ 0.3

+ 2.1

0

Average for 10 experiments .

Work experiments:

1 experiment with E. 0.

2 experiments with J. F. S. .

26

4
6

2290

3922
3583

2305

3928
3552

+ 15

+ 6
-31

+ 0.7

+ 0.2
-0.9

Average for 3 experiments . .

Average for all rest and work
experiments withspecialdiet

Average for all the above ex-
periments

10
36
143

3719
2687
3481

3702
2695
3481

-17

+ 8

0

-0.5

+ 0.3

0

336

LECTURE XV.

As the values show, it is evident that the law of the conservation of
energy holds with surprising exactness for the whole animal organism. It
was found possible to obtain such a close agreement between the sum of
the amounts of energy introduced into the body and that produced by
combustion within the organism, only by extending the experiment
through quite a number of days.

This brings us to the important question as to whether the chemical
energies introduced into the body by the individual nutrients are all
equivalent, or, in other words, whether it is immateiial in what form the
animal organism receives its chemical energy. After considerable investi-
gation Max Rubner * was able to show that the different organic nutrients
could replace one another in amounts corresponding approximately to
their relative heat values. This principle comprises the Law of Isody-
namics. According to this law we can represent each substance used as
food in a common unit and give it a calorific value. Thus, for example,
100 grams of fat are isodynamic with the following weights of

Syntonin . .
Dry meat . .
Starch . . .
Cane-sugar .
Grape-sugar

213
235
229
235
255

Strictly speaking, the Law of Isodynamics only applies to fats and
carbohydrates. It fails with the albumins. These, to a certain extent,
are absolutely necessary for the animal organism. It is indeed possible
to keep a dog alive for a long time on albumin alone; i.e., the albumin
itself may be looked upon as isodynamic with the fats and carbohydrates.
It is, however, as we shall see later on, impossible to nourish a dog on
fats and carbohydrates exclusively, even when more than sufficient calo-
rific units are provided. Starvation metabolism begins in the absence
of albuminous material; i.e., the animal draws on its own body albumin,
for which it has no substitute.

Studies on metabolism have shown how much nourishment is required
for the maintenance of a definite organism, and how to express this require-
ment in terms of calories. We shall consider these relations more in detail
later. Here we shall only state, that the exact formulation of the total
metabolism obtained by considering the foods solely as combustible
material, although of great importance for the entire conception of meta-

' Cf. Max Rubner: Die Gesetze des Energieverbrauches bei der Emahrung. Franz
Deuticke, Leipsic and Vienna, 1902.

FATS, CARBOHYDRATES, AND ALBUMINS. 337

holism, by no means tells the whole story. The calorific values serve
merely as a skeleton, and give us an outline of the changes which take
place in metabolism. These changes are always to be traced back to the
individual cells. It is not the foods as such which determine in general
the metabolism, but the cells themselves. These, naturally, require a
certain amount of energy. We shall see later on, that metabolism varies
in different individuals, and that the consumption of material, to a large
extent, is regulated by the functional activity of the separate organs.
The same work — e.g., a definite amount of muscular work — • will require
a greater quantity of energy the first time it is performed than when it is
repeated. By practice, the organism adapts itself to its requirements.
It learns how to perform a given amount of work with the least expenditure
of energy. We must call attention even here to this fact in order to show
that experiments in metabolism, and especially experiments dealing with
the energy required for a definite amount of work, are not likely to give
true values, unless they be carried out through an extended period of
time. It will only then be possible to compare the fluctuations and
irregularities of the separate daily periods, and it is only in this manner
that we shall be able to obtain values which will be comparable with others
which have been secured under different circumstances. In practical
work, as we shall see later, we do not study the fats, carbohydrates, and
proteins by themselves, but make use of those mixtures which are present
naturally in meat and vegetables. The impracticability of laying too
much stress upon the calorific values is very well shown by the significant
discovery that the whole work of the digestive glands, and consequently
digestion itself, is dependent to a great extent upon the nature of the
ingested food material. It is only by combining the knowledge gained
from investigations on the transformation of energy with that obtained
in the study of cell activity that we are enabled to get a complete conception
of metabolism in general.

We are first of all interested in the questions: What relations do the
foods bear to one another, and what proofs do we have that certain organs
are able to perform definite functions with all three classes of nutrients ?
Let us first take up the last question. In discussing the carbohydrates,
we have already drawn the conclusion, from many experiments, that they
form an exceptionally important source of muscular activity. Now, are
the muscles also capable of performing their functions by utilizing the
energy contained in representatives of the two other kinds of organic
food, the fats and proteins?

The fact that protein may serve as a source of muscular energy was
proved by Pfliiger.' For over seven months he fed a dog exclusively on
meat which contained only small amounts of fat and carbohydrates. — in
fact, not enough to satisfy the requirements of the heart's work. Pfliiger.

' E. Pfluger- Pfliiger's Arch. 50, 98, 330, 396 (1891).

338 LECTURE XV.

furthermore, made this dog do heavy work for considerable lengths of
time. The dog, therefore, had to perform all of its muscular work at the
expense of protein. This proves that protein can also serve as a source of
muscular energy. Under normal conditions, i.e., with a mixed diet the
muscle cells will first make use of the carbohydrates as a source of energy
and, if this is exhausted, then attack the protein.

A much discussed question is the value of the fats as a source of muscular
energy. Chauveau ' and others took the stand that fat, as such, can in
no case be utilized as a source of energy by the muscle cells for the per-
formance of work. The fat in every case must first be transformed into
sugar before it can be used by the muscle cells. According to this assump-
tion, the value of fats for the production of muscular energy could not be
larger than that corresponding to the quantity of sugar which this amount
of fat can form. Now 1 gram of fat is isodynamic with 2.56 grams of
dextrose when the heat units of both are taken into consideration.

If we assume that the fats, before they can be utilized, must be changed
into carbohydrates, it follows that 1 gram of fat would correspond to 1.6
grams of carbohydrate, if the fat is oxidized directly to sugar. We might
expect to be able to determine by direct experiment how much of the
potential energy in fats the body can transform into muscular force. This
has not yet been satisfactorily accomplished. If we feed ^n animal with
a mixture of albumin, fat, and carbohydrate, the calorific value of which
we know exactly, we are unable to decide by which part of the energy the
animal organism performs its different functions. We do not know whether
such a selection actually does take place in the case of a mixed diet, or if
it is not more probable that the organism takes all of the energy presented
as such, and uses it for all of its functions. Atwater^ justly calls attention
to the fact that we have no means of differentiating internal from external
muscular work. We must also remember that in every case only a part of
the energy used for accomplishing work is shown by the work performed.
A large part of this energy is transformed into heat. We can obtain an
idea indirectly of the value of fats as a source of muscular work, if we regu-
late the conditions of the experiment so that an economical utilization
of the available energy is guaranteed. If in the food only barely enough
energy is supplied to the body to meet its requirements, or even less than
enough, we would expect it to utilize all of the available energy in the
most economical manner. Atwater has carried out such experiments.
Particular stress is laid on the fact that the albumin in the food must be
limited as much as possible in order to compare the fats and carbohydrates.
Atwater, therefore, used only about as much protein in severe muscular
work as he found necessary to maintain the nitrogen equilibrium in a

' A. Chauveau: Cf. Compt. rend. 121, 26, 91 (1895); 122, 429, 504, 1098, 1163, 1169,
1244, 1303 (1896); 123, 151, 283 (1897).

' Atwater: Ergeb. Physiol III, Abt. 1, p. 497 (1904).

FATS, CARBOHYDRATES, AND ALBUMINS.

339

previous " rest " experiment. This amount of albumin, with small quan-
tities of fat and carbohydrate, was the starting ration of the " work "
experiment. In the principal test, in one case a considerable amount of
fat (butter, cream) was added, while in another experiment an equivalent
amount of carbohydrate (milk-sugar and cane-sugar) was employed. The
total energies received was somewhat less than the organism required; that
is, some of the body substance was attacked. These experiments estab-
lished the values of fats and carbohydrates as sources of muscular energy
in two directions. In both cases the energy in the form of albumin and the
total energy received were the same, the only differences being the predomi-
nance of fat in the one case, and of carbohydrates in the other. The
external work was likewise the same in both cases. If the total energy
utilized for the production of a definite amount of work was the same for
a fat as for a carbohydrate diet, the fact would be established that fats
and carbohydrates have the same value as a source of muscular energy.
In the next place the amount of energy abstracted from the body itself —
the quantity of energy received in the food was, as already stated, not
quite enough to satisfy the requirements — - must be a measure of the
relative value of a diet in which either fat or carbohydrate predominates.
If equal quantities of body substance were used up in both cases, we
would have a further support of the equality of carbohydrates and fats
as sources of muscular energy. The following taljle will give us an idea
of the results obtained by Atwater in his experiments.

REL.\TIVE V.A.LUES OF FATS AND CARBOHYDRATES IX THE FOOD
FOR THE PERFORMANCE OF MUSCULAR WORK.

Name, Nature of the Experiment.

Time.

Energy in
the Food.

Energy
Equiva-
lent to
External
Muscular
Work.

Energy of
the Oxi-
dized Ma-
terial.

Energ.v
Equiva-
lent to the
Gain { + )
or Loss
(-) of

Days.

Calories.

Body Sub-
stance.

No. 40 J. C. W. carbohydrate diet

No. 41 J. C. W. fat diet

No. 44 J. C. W. carbohydrate diet

No. 43 J. C. W. fat diet

No. 47 J. C. W. carbohydrate diet

No. 46 J. C. W. fat diet

No. 53 J. C. W. carbohydrate diet
No. 52 J.C. W. fat diet

4
4
4
4
4
4
3
3

4180
4150
4602
4496
4366
4478
5132
5120

518
522
571
548
562
551
587
607

5251
5304
5125
5155
5173
5193
5104
5309

-1071
-1154

- 523

- 659

- 807

- 715
+ 28

- 189

Average of 4 experiments with
carbohydrate diet

Average of 4 experiments with fat
diet

15
15

4532
4524

558
554

5167
5236

- 635
712

340

LECTURE XV.

These experiments show somewhat higher values for the carbohydrates
than for the fats. It is questionable whether this is always true, for
Atwater also published the results of some experiments in which this was
not the case. That, as a matter of fact, the fats are to be considered as
direct sources of muscular work, without requiring any preliminary con-
version into carbohydrates, seems apparent ; for if we assume that the fats
are first transformed into carbohydrates, there will be a loss of energy
during their oxidation. In such a transformation, 36 per cent of the
potential energy of the fat would become free energy. Now 1 gram of
animal fat produces 9.50 calories, and 1 gram of cane-sugar .3.96 calories.
By the combustion, in the bomb calorimeter, 10.53 grams of fat and
25 . 25 grams sugar are required to produce 100 calories. The same number
of calories would, of course, be liberated in the body during complete com-
bustion. If the carbohydrates alone were the source of muscular energy,
36 of the 100 calories from the 10.53 grams of fat would not be utilized.
These would be set free in the body during the transformation of the fats
into carbohydrates and appear as heat. The ratio of the 10.53 grams
fat to the 25.25 grams carbohydrate would be 64 : 100. This, as a
matter of fact, is not the case, as the following table shows. In it the
relative values of a carbohydrate and of a fat diet, as shown by the above
experiments, are compared; in one case the amount of energy transformed
per day is given in calories, and in the other the percentage of utilization
of the fat diet is compared with that of the carbohydrate diet, and fat
alone is compared with carbohydrate.

PERCENTAGE UTILIZATION OF ENERGY.

Experiment.

Energy from Fat Diet

Compared with ttiat of

Carbohydrate Diet.

Energy in Fat Com-
pared with that in
Carbohydrate.

Experiments No. 40 and 41

Experiments No. 43 and 44

Experiments No. 46 and 47

E.xperiments No. 52 and 53

Per cent.
99.2
96,8
98.3
97.7

Per cent.
98 3
92 8
96.2
94.8

Average

98.0

95.5

Instead of the theoretical ratio of 64 : 100 we find that the fat stands
to the carbohydrate in the proportion of 95.5 : 98.0. Thus, it is evident,
unless we choose a far more complicated explanation, that the energy
which the body receives in the form of fat is a direct source of muscular
energy, and that a preliminary transformation of fat into carbohydrate
does not take place.

FATS, CARBOHYDRATES, AND ALBUMINS. 341

If we apply these relations to the metabolism of a diabetic, we will
appreciate the great derangement of his energy economy. In the severe
form of this disease, the organism loses not only the greater part of the
energy of the carbohydrates, but also the energy required to transform
fat or protein into sugar; and as a part of the sugar so formed is eliminated
by the system, the loss becomes a double one. The fact that the diabetic,
whose blood and tissues are saturated with sugar, and who is already
greatly injured as regards the economy of energy by means of the loss he
suffers because of his inability to consume sugar, even prepares more
sugar from the other nutrients, only to eliminate it eventually as such,
shows us that the assumption that diabetes is only a simple derangement
of carbohydrate metabolism does not satisfactorily explain the disease.
Up to the present time the most prominent symptom, that of glucosuria,
has dominated the entire investigation of problems concerning diabetes,
and it is very probable that this is the reason why the disease, as a whole,
is so little understood.

We have intentionally gone somewhat into detail concerning these
relations, because we are unable to follow any other function so exactly
as that of muscular work. All the other organs are only indirectly acces-
sible to our observation. We do not know whether they also are able to
utilize all three of these materials, fat, carbohydrate, and protein, in like
manner as sources of energy. O. Cohnheim ' has recently performed an
experiment in this direction. He tried to decide whether the digestive
glands obtained their energy requirements mainly or exclusively from pro-
tein. As we shall see later on, it is possible to stimulate the digestive
glands without introducing into the alimentary tract food which would
then participate in the metabolism. Cohnheim made an oesophageal
fistula in a dog according to the method of Pawlow, and after a period of
fasting fed the animal. The food eaten by the animal fell out through the
fistula tube at every swallow, so that no nourishment was actually received
by the dog. Not only are the salivary glands stimulated by this " fictitious
feeding," but the stomach also. On account of the acid gastric juice
passing from the stomach into the duodenum, the pancreatic gland also
begins to secrete its fluid. By estimating the amount of nitrogen present
in the urine on days of fasting and those on which the fictitious feeding
day took place, Cohnheim succeeded in showing that the activities of the
digestive organs were without influence upon the transformation of albu-
min. No increased elimination of nitrogen took place. This does not by
any means prove that the digestive glands do not work with albumin.
It is possible that, while the digestive glands are decomposing albumin,
an equivalent amount of albumin is being " spared " in some other part
of the body. Such an assumption becomes all the more plausible when

> O. Cohnheim: Z. physiol. Chem. 46, 9 (1905).

342 _ LECTURE XV.

we remember that the animal was fasting, and that such an organism is
as economical as possible with its subsistence. On the other hand, it is
possible that all three nutrients, fat, carbohydrate, and albumin, are drawn
upon iis sources of the energy required for the work of the glands, and that
the consumption, of albumin especially, was so small, that it did not
change the nitrogen content of the urine enough to be shown by our
present methods of analysis.

We have already stated that the carbohydrates are also to be looked upon
as a source of heat. It is possible to cause glycogen to disappear by merely
chilling an animal. Albumin may also act in this way as a source of heat.

.4t all events, the discovery that the fats act as direct sources of mus-
cular force, proves that the nutrients stand in intimate relation to one
another. They replace one another partly by being transformed and
partly by reason of their calorific values. This, however, does not by any
means include all of the relations existing between the individual nutrients.
As we have already seen, it is possil:>le to keep a dog alive on meat alone.
In this case the organism must obtain all of its requirements from the
albumin (aside from the small amounts of fat and carbohydrate contained
in the meat). If the quantity of meat is insufficient, the animal must
draw upon its stores of fat and carbohydrate. It is a simple matter to
determine the amount of meat necessary to just satisfy the energy require-
ments. If we exceed to the slightest degree the quantity of lean meat
which is necessary to keep the metabolism in equilibrium, there will be
no accumulation of albumin, but, on the contrary, there is an increased
decomposition of albumin as a consequence. An accumulation of albumin,
i.e., an increased amount of albumin in the cell material, may indeed be
brought about by a liberal and long-continued feeding of albuminous
material.' It has been shown, however, that the animal organism con-
stantly seeks to maintain its albumin content, and consequently the func-
tional condition of its cells, at a constant level. As soon as the diet ceases
to contain the excess of protein, the accumulation of albumin in the cells
quickly disappears. The previous equilibrium in the economy of the
individual cells will be reestablished.

If we make an animal go hungry, the elimination of nitrogen continues,
and this is also true when an abundance of fat or carbohytlrate is fed to
the animal. The albumin, therefore, is not entirely replaceable, although
it is possible to reduce the destruction of albumin by means of fats. This
fat may come from the food or from the body itself. It is possible by
means of fat and albumin to establish a new nitrogen balance; i.e., to

• More recent experiments make it seem doubtful whether the nitrogen retention
and deposition of albumin correspond to one another. Great caution should be used in
the estimation of the nitrogen balance in this direction. Cf. E. Abderhalden and
Bloch: Z. physiol. Chem. 53, 464 (1907J.

FATS, CARBOHYDRATES, AND ALBUMINS.

343

determine anew the quantity of albumin which is absolutely necessary to
prevent injury to the albumin content of the organism. This amount of
albumin will be much less than is required in a diet of albumin alone. If
we feed an animal a definite quantity of fat and albumin, it will be possible
to reduce the albumin requirement more and more by increasing the pro-
portion of fat in mixture. We finally reach a minimum amount of pro-
tein, and if we attempt to replace it by fat, we cause the body albumin
itself to be attacked. This limit varies with different animals and at
different times with the same animal; in every organism it is dependent
upon the condition of the body, and, above all, upon the fat in the body at
the time of the experiment. A fat animal will permit more albumin to be
replaced by fat than will a lean one, for the former can contribute from
its own supply of fat. On the other hand, by feeding fat we have a means
of causing an accumulation of albumin. We can spare albumin not only
with fat, but also with carbohydrates. By their assistance also, provided
sufficient albumin is supplied, the albumin content of the body may be
increased. Voit,' who performed experiments in this direction, came to
the conclusion that the fats and carboh3fdrates did not have an equivalent
effect in causing an accumulation of albumin. Carbohydrates are more
efficient as " albumin sparers " than are the fats, as is shown by the follow-
ing table:

Food.

Meat.

Gain (+) Loss (-)

Meat.

Fat.

Carbohydrate.
Starch. Sugar.

Transformed into Body
.\lbumin.

in Body Albumin.

500

250

558

- 58

500

300

466

+ 34

500

200

505

- 5

800

250

745

+ 55

800

200

773

+ 27

2000

200-300

1792

+ 208

2000

250

1883

+ 117

Atwater - has recently determined accurately the comparative values of
carbohydrates and fats as sparers of albumin. The fact that proteins are
distinguished from the other nutrients by the amount of nitrogen which
they contain, makes it easy to carry out the experiment. By simply
comparing the amount of nitrogen in the food with that of the urine, we
can at once get an idea as to how much albumin has been decomposed.
The nitrogen content of the fseces tells us approximately how much albumin
has not been absorbed. We speak of a " nitrogen equilibrium " when the
amount of nitrogen ingested in the form of food is equal to that con-
tained in the urine and in the faeces. If the latter is greater than the

' Carl Voit: Hermann's Handbuch der Physiologic, 6, 14.3 (1881). ' he. cit.

344

LECTURE XV.

amount introduced, it shows that alljumin from the body has been decom-
posed. Conversely, if the nitrogen eliminated is less than that ingested,
then we are justified in concluding that albumin has been accumulated.

The following table will give a summary of some of Atwater's experi-
ments:

w

o 1

jl

40
41
44
43
47
46
53
52

1

fj -a
1 =

<

k ^ .

Nitrogen

Kind of Experiment.

Sub-
ject.

III

1

s

.1

-i-T

Calories.

Grams.

Work experiments:

Carbohydrate

Fat

Carbohydrate

Fat

Carbohydrate

Fat

Carbohydrate

Fat

J.C

.W

4
4
4
4
4
4
3
3

4180
4150
4602
4496
4366
4478
5132
5120

518
522
571
548
562
551
587
607

17.1
16.9
17.8
17.1
17.4
17.0
17.9
17.7

2.2
1.5
2.6
2.0
2.7
1.8
2.3
1.6

17.1
20.3
17.3
19.1
16.3
16.1
15.4
16.4

-2.2
-4.9
-2.1
-4.0
-1.6
-0.9
-F0.2
-0.3

Average for the experi-
ments with carbohydrates

Average for the experi-
ments with fat

15
15

4532

4524

558
554

17.5
17.1

2.5

1.7

16.6
18.1

-1.5
-2.7

From these experiments we find, in agreement with those of Voit, that
carbohydrates as well as fats act as sparers of albumin, under the given
conditions. The fact that in the carbohydrate experiments there was
invariably more nitrogen in the fteces than in the experiments with the
fats, is explained by the nature of the food. Vegetables predominated
in the former case, and meat in the latter. We shall see later, that the
protein in vegetables is utilized to a less extent than that of meat. If we
subtract the nitrogen in the faeces from the total nitrogen of the food, we
shall obtain the quantity of nitrogen which the organism has evidently
utilized. If we insert these values in the above table, we shall find that
less nitrogen was available when carbohydrates were eaten, than when
fats predominated in the food. Nevertheless, there was less nitrogen
present in the urine in the former case than in the latter, although the
difference was not great.

These experiments still leave one question unsolved: How do the
fats and carbohydrates act as sparers of albumin, when they are eaten
together? In the above experiments, the food at one time contained
protein and fat, and at another time protein and carbohydrate. Under
ordinary conditions all three kinds of nutrients, fat, carbohydrate, and
protein, are available to the organism. Tallquist ' has, therefore, studied

' F. W. Tallquist: Arch. Hyg. 41, 177 (1902).

FATS, CARBOHYDRATES, AND ALBUMINS. 345

the problem from this standpoint. During one period, the food contained
16.08 grams nitrogen, 44 grams fat, and 466 grams carbohydrates; and
in a parallel experiment it contained 16.08 grams nitrogen, 146 grams fat,
and 250 grams carbohydrates; each gives the same number of calories
(2867 and 2873 calories). In both experiments practically the same
nitrogen balance was reached. It appears that under these conditions
the carbohydrates are isodynamic with the fats in respect to acting as
albumin sparers. Landergren ' explains the greater sparing of albumin in
an exclusively carbohydrate diet, as compared to a diet of fats alone, by
the assumption that the animal organism constantly requires sugar; and
inasmuch as he does not believe fat can be changed into sugar, a part of
the albumin must be utilized, in the case of a fat diet, for the formation
of sugar. This part of the albumin is spared, if the food contains carbo-
hydrates, so that the organism then has more albumin at its disposal for
its remaining functions. The total albumin may be utilized as such, when
sufficient fat and carbohydrate has been added to the diet. This expla-
nation is at present only an hypothesis.

The results as a whole, which have been obtained in studying the rela-
tions of the carbohydrates and fats to the transformation of albumin,
show that we may conclude with a great degree of probability that the
Law of Isodynamics holds for these nutrients among themselves. Both
are able to spare albumin to about the same extent. The fact that the
feeding of carbohydrate alone, or fat by itself, should have shown a differ-
ence in favor of the former, may be explained by the assumption that this
Law of Isodynamics is not an absolute one; i.e., the chemical composition
of the foods undoubtedly plays an important part.

We have already mentioned the most important relations existing
between the nitrogen-free nutrients; i.e., the fats and carbohydrates.
We have shown that with fats the muscles are able to perform their work
just as well as with carbohydrates. On the other hand, we know of
experiments which prove beyond question that carbohydrates can replace
fats; in fact, according to the Law of Isodynamics. If all nourishment
is withheld from an animal, it will draw on its own body, not only attack-
ing its own protein, but especially the fat deposits. If we substitute for
the fat which would be used up in this way during starvation the same
quantity of fat in food, we find that a complete replacement follows;
i.e., the animal does not touch its fat reserves. The same effect can be
obtained by substituting for the fat an isodynamic amount of carbohy-
drate. This is shown by the following table, which gives a summary of
some of Atwater's experiments.

The loss of body fat was at one time greater with a carbohydrate diet,

' E. Landergren: Skand. Anh. Physiol. 14, 112 (190.3).

346

LECTURE XV.

and at another time greater with a fat diet. On an average it was lesa
with the carbohydrate diet. Apparently sugar is a better sparer of body
fat than fat itself. The difference is, however, very slight, and we may
conclude from the experiments that isodynamic quantities of fat and
carbohydrates are equivalent in this respect.

Available

in the

_ >,

-

Gain (-H) or Loss (— )

Food.

Is

W ij

Kiml of Experiment.

Sub-
ject.

No.

Q

E
O

3 ■

It

o .S

c "5

•5^

£

1

1 S

c

c

° C

o

w

1

■3 S

40

Q
4

1
17.1

ffi "

MO

9J d

Calories.

Grams.

!•-

Carbohydrate . . .

J.C.W.

4180

5251

518

-13.6

- 77

-104.2

-994

Fat . . . ^. .

«

41
44

4
4

16.9

17.8

4150
4602

5304
5125

522
571

-30.6
-13.1

-173
- 74

-102.8
- 47.1

-981

Carbohydrate

-449

Fat

"

43

4

17.1

4416

5155

548

-25.0

-141

- 54.3

-518

Carbohydrate

47

4

17.4

4366

5173

562

-10.1

- 58

- 78.5

-749

Fat

"

46

4

17.0

4478

5193

551

- 5,6

- 32

-71.6

-683

Carbohydrate

"

53

3

17.9

5132

5104

587

+ 1.3

-1- 8

+ 2.1

+ 20

Fat .... .

"

52

3

17.7

5120

5309

607

- 2.1

- 12

- 18.6

-177

Average for the ex-

periments with

carbohydrates . .

15

17.5

4532

5167

558

- 9 5

- 54

- 60.7

-581

Average for the ex-

periments with

fat.s

15

17.1

4524

5236

554

-16.7

- 95

-64.7

-617

The question that next arises is with regard to the way in which carbo-
hydrates and fats behave when they are fed simultaneously. It would
seem possible that they would be decomposed equally, and the liberated
energy utilized, sometimes for one purpose and sometimes for another.
It is also conceivable that one substance may be used up more rapidly
than the other. This is a very hard problem to decide. We may determine
the amount of nitrogen and carbon eliminated in the urine and faces, and
deduct from the total amount of carbon that corresponding to the protein
(as indicated by the nitrogen value). The carbohydrates and fats would
1)6 represented by the rest of the carbon. It would be possible to decide
which nutrient gave rise to the greater part of the carbon dioxide formed
if we knew the amount of oxygen which was consumed at the same time.
If carbohydrates were exclusively oxidized, the ratio of the volume of oxygen
taken up, to that of carbon dioxide produced, would be equal to 1. The
ratio of CO2 : O2 is called the " respiratory quotient." It is only 0.71 in
the case of the fats. Experiments which have been made in this directioa

FATS, CARBOHYDRATES, AND ALBUMINS.

347

indicate that the carbohydrates are attacked immediately after absorption
from the intestine, thus sparing the body fat. We must not forget, with
regard to such experiments, that the conclusions drawn are for the most
part indirect. They are not conclusive. A comparison of the general
metabolism on the basis of income and outgo alone, must necessarily lead
to a one-sided decision. The results of physiological-chemical investi-
gations must not be left out of consideration, and the finer details of the
work must not be forgotten in studying the coarser outlines of metabolism
as a whole. We have seen that the metabolism of carbohydrates in all
of its phases is an extremely delicately regulated process. Sugar reappears,
after absorption, in the liver, deposited in the form of glycogen. This
does not vanish so easily. It is extremely difficult to obtain an animal
which is free from glycogen. At all events, our knowledge regarding the
transformation of the carbohydrates in the tissues is altogether opposed to
the assumption that they are rapidly burned up after absorption, even
during work. We know it is true that the muscles evidently prefer to
utiUze the energy from carbohydrates in their performance of work, but
this very fact prevents us from believing that the organism consumes this
valuable material in order to save the fats.

Studies on metabolism have shown us that there is a difference in the
behavior of the protein in food and that of fats and carbohydrates,
which are themselves very similar in their behavior. We have already
stated that by increasing the amount of protein in food, there is an in-
creased metabolism. This, however, is not the case, or at least not to the
same extent, if we increase the quantities of fat or carbohydrate, as the
following experiments, carried out by M. Rubner, show:

Income.

Total Metabolism in Calories.

Day.

Nitrogen in
Grams.

Fat in Grams.

Carbohydrate
in Grams.

In Calories.

Absolute.

Per Kilogram
of Body

Weight.

2
3
4
5
6
7
8

56.8

167

ill

1513
1536
1446

969
1072
947
963
922
982
977

40.2
44.8
39.9
40,9
39.6
42.3
42.1

2, 4, 6, and 8 are fasting experiments. In them 40.4 calories per kilo-
gram body weight is the average metabolism. The addition of albumin
causes an increase in the number of calories amounting to 11.9 per cent;
the addition of fat, 1.2 per cent; and that of carbohydrates, 4.2 per cent.

348 LECTURE XV.

The figures show that the number of calories in each case were about the
same in the three experiments. If such experiments are carried out
unaccompanied by muscular work, the difference between the effect of
albumin, on the one hand, and that of the carbohydrates and fats, on the
other, towards the entire metabolism, becomes more marked.

We have now finished all that we care to say with regard to the mutual
relations existing among the three most important organic nutrients.
We have discussed two ways in which they may replace one another. On
the one hand, it is possible by means of a chemical transformation for one
nutrient to be converted into another, and, again, the replacement may
be merely one of calorific values, without any such transformation being
necessary. The latter method of one food replacing another is undoubtedly
of the greatest importance in the whole economy of metabolism. It
represents the greatest possible utilization of the available energy, and
guarantees the satisfactory maintenance of the entire metabolism, even
when one of the nutrients is not momentarily available. Albumin is an
exception. It is only in part replaceable. If the organism is starving,
it tries to preserve its albumin by consuming first the fats and car-
bohydrates available, thus protecting its tissues against severe injury.
The former condition of the body is therefore quickly regained as soon as
food is eaten again. It is only when the organism draws upon its own
protein for the main supply of the required energy, that the end is near.
It is of great interest to note, that the reserve materials held in store by
the organism, and drawn upon during starvation, likewise enter into metab-
olism strictly in accordance with their calorific values. As soon as the
body substances are called upon to act as combustible material, they also
follow the Law of Isodynamics.

LECTURE XVI.

INORGANIC FOODS.

Importance of Inorganic Substances as Building Material of
THE Cells and Tissue. — Water, Salts.

All of the food-stuffs which we have studied up to the present, are those
by means of which the animal may obtain chemical energy. The con-
ception of a food is, in fact, closely related to this property. We recognize,
however, a group of compounds indispensable to the organism which it
always receives with its food, but from which it can obtain no chemical
energy. We refer to water and inorganic salts. Thus far we have con-
sidered the foods solely with regard to their value as sources of kinetic
energy. We must not forget, however, that the organism is constantly
wearing out its cell-material; in fact, individual cells may even be entirely
thrown off, only to be regenerated and built up anew. Such processes
are particularly noticeable in the case of growing organisms. In such
cases the new cells formed in place of the old ones are often of larger
dimensions. Yet, it must not be thought that the fully developed
organism retains its cellular condition unchanged. At present we are not
in a position to explain fully the metabolism within the individual cells.
We have no means of knowing how long a single cell may live; we do not
know how long it can exercise its function with the same material. All
that we can say is that there are certain processes visible to the eye which
give indication of a continuous breaking down and building up of cells.
We know that hair, feathers, scales, etc., undergo constant changes,
processes which take place in some species of animals very slowly, but
continuously; while in other cases, as with the shedding of feathers in the
case of birds, and the changing of skins with reptiles and amphibia, such
processes take place within a relatively short time. .Again, we know of
the constant change in the cells of the epidermis, and in the cells of the
mucous membrane. Similarly we know that there is a continual loss of
material involved in the exercise of the function of numerous glands. In
this connection we need merely mention the glands of the skin, — the
sebaceous and sweat glands, — the salivary glands, and the numerous little
mucous glands of the respiratory and digestive membranes. The same

349

350 LECTURE XVI.

is true of all the glands taking part in the digestive processes, beginning
with those of the stomach and the mucous membrane of the alimentary
canal, on to the large digestive glands of the pancreas and liver. In all
such cases the organism suffers a constant loss of material. Again, the
organism constantly requires the presence of salts, and water to flush out
the waste material through the kidneys. Further, we have to consider
the specific tasks of the single cells and cell-groups by means of which
definite products are developed which play an important part in the metab-
olism of the organs, whether it be a ferment, or some other product such
as adrenalin, which is formed in the suprarenal gland.

Furthermore, the fact that there is evidently a lively breaking down
and building up even in tissues which we would scarcely expect to par-
ticipate in active metabolism, is shown to us by a histologic study of the
bones, which show evidence of a continual exchange of their building
material. From the field of pathology, we find that the building up of
the nerves and their restitution under some conditions may assume con-
siderable dimensions. This is shown, for example, in the case o/ hyper-
trophic activity, which appears as soion as there is an additional require-
ment placed upon a certain organ, whether because of the fact that it
must act as a substitute for another, or whether because its work becomes
increased in some other abnormal way, as, for example, in the case of the
heart in certain kinds of heart trouble. On the other hand, in conva-
lescence after certain fevers, particularly typhoid, we find a sudden reju-
venation of the sunken cell-energy. Each cell takes up the building
material from the circulating nourishment, and this is particularly true of
albumin, which in a certain sense determines the functional activity of
the cell. In a short time the organism is renewed. The loss of albumin
which the body has experienced during the disease is quickly compen-
sated. The old equilibrium in the economy of the cells is again estab-
lished. Again, a sudden increased production of cell-material takes place
after some local irritation. Thus we find that the organism concentrates
a great number of leucocytes at an infected point, and finally perhaps
large masses of pus are formed, all at the cost of the nourishment and the
material composing the organs. On the other hand, sometimes we find
the organism throwing off considerable quantities of exudate, as, for
example, in pneumonia, which again uses up large amounts of material.
On the other hand, if we consider the continual variations in the number
of red and white corpuscles, and the variations in the lymphocytes, we
obtain the impression that the cell-material of the fully developed organism
is never at rest. We know practically nothing concerning the quantitative
relations involved in all such changes. We do not know whether the
material in the old cells is used to some extent in building up new ones,
or whether the new cells are entirely formed from new material. We do

INORGANIC FOODS.

351

not know whether the individual organs can effect an exchange of materials,
or whether the cells of one group can utilize the waste material of another.
The interesting studies of Miescher ' on salmon give us some information
in this direction. Previous to spawning, these fish migrate from the sea
into fresh water, for example, into the Rhine. From the time that these
fish reach the river up to the time that the eggs are laid, they take no
nourishment. This fact was known to Barfurth - and to His.' Meischer
estimated that the majority of the salmon remained in the Rhine for from
six to nine and one-half months, a smaller number stayed up to twelve
months, while some were there as long as fifteen months. During all of
the time that the fish remains in fresh water, nothing is eaten. The intes-
tines are always found empty; and, indeed, Miescher established the fact
that the digestive glands during this period do not yield any active juices.
A series of marked changes take place in the appearance of the fish during
this period. When the salmon first reaches fresh water its organs of regen-
eration are quite undeveloped. Being provided with a powerful dorsal
musculature, it is able to stem the most rapid currents in the Rhine. On
comparing such a fish with one that is taken just before the spawning
time, it seems scarcely possible that they are the same kind of fish.
The large dorsal muscle has become shrunken; the sexual organs, on
the other hand, have become enormously developed. There is a parallel-
ism between the two changes. Miescher observed, for example, that the
weight of the ovary increased from 9.4 grams to 15 grams, while simul-
taneously there was a diminution in the dry substance and in the albumin
content of the dorsal muscles as shown by the following average values:

Length in
Milli-
meters.

Weight in
Grams.

Weight of
Ovary in
Per cent
of Body
Weight.

Composition of Dor-
sal Muscle.

Per cent
Albumin.

Per cent
Dry Sub-
stance.

March

July and August

November and December ...

872
886
879

9305
8953
7428

0.061

4.78

18.45

17.5

13.2

33.6

26.8
IS. 5

The albumin lost by the muscles is evidently utilized in building up the
sexual glands — in one case the eggs, and in the other the sperm cells.

' Die histochemischen und physiologischen Arbeiten von Friedrich Miescher, vol. ii.
Leipsic, 1897. Pp. 116 et. seq.

' Troschel's Arch. Naturgeschichte, Jg. xli, I, 122 (1875).

' Untersuchungen iiber das Ei und die Eientwicklung bei Knochenfischen. Leipsic,
1873.

352 LECTURE XVI.

Direct observation confirms this. Microscopic examination of the ovaries
and of the testes shows that most active processes of growth and trans-
formation are taking place. On the other hand, the large dorsal muscle
exhibits all the signs of a far-reaching release of its stored material
and of even the contents of its cells, the muscular fibers. Everything is
not given up, but as much of the material as can be spared is transformed,
leaving enough behind so that subsequently when the salmon returns to
the sea the muscles may be regenerated. It is the large dorsal muscle
which entirely provides the material for the changes taking place in the
body of the starving fish, whereas the remaining muscles undergo no
change that indicates in any way a migration of material. It has never
been satisfactorily explained just how this migration takes place. Miescher
describes the appearance of small drops of fat between the muscle fibrils.
The amount of these drops may become so great that the whole muscular
fiber becomes opaque. It is obvious that in this way preparation is
being made for a migration of fat. Besides albumin and fat, the muscle
must give up phosphates which in the formation of eggs evidently become
a part of the lecithin. The other salts and substances, such as choles-
terol and the nuclein substances required to form the nuclei of the eggs,
must likewise be obtained from the dorsal muscle. It is evident, there-
fore, that the migration of substance here attains large dimensions.
Undoubtedly a study of this interesting biological phenomenon by modern
methods would give us considerable insight into the extent of the syntheses
capable of being carried out in the organism. Every supply from the
outside is cut off. The entire sexual products are formed from material
taken from the dorsal muscle. It is clear that a comparison of tJie amount
of lecithin, nucleic acids, etc., contained in this muscle with those of the
sexual products, would give us a good idea of the metabolism and of the
chemical processes involved.

These observations permit us to draw certain conclusions concerning
metabolism during starvation.' If every source of food supply is cut off
from an animal, then the»organism turns to its own body for nourishment.
Carbon dioxide is constantly being eliminated and oxygen absorbed, and
likewise the chemical composition of the urine gives unmistakable proof
that combustion is continually taking place, which in warm-blooded
animals suffices not only for the performance of mechanical work, but also
for maintaining the temperature of the body. If we compare the relative
proportions by weight of the separate organs of a starving animal with
those of one that is well nourished, it is at once apparent that the different
organs do not participate equally in the metabolism. Thus the nervous
system and the heart show but slight, if any, changes in their composition

' S. M. Lukjanow: Z. physiol. Chera. 13, 339 (1889). C. Voit: Z. Biol. 31, 510 (1894).
A. Hermann: Pfluger's Arch. 43, 239 (1888).

INORGANIC FOODS. 353

as regards both organic and inorganic matter. Evidently these organs
which are so indispensable to life are maintained at the expense of the
less vital tissues. In such cases there is a continuous transportation of
material from one tissue to another. The fact that one organ, which
during starvation will normally lose material to a considerable extent,
may under other conditions, i.e., when its function is of especial impor-
tance to the whole organism, be kept in full activity at the expense of
other organs, is shown by an observation made by Pfliiger ' that the liver
of dogs after extirpation of the pancreas, in spite of the resulting glucosuria,
did not diminish in weight, whereas all the other organs — excepting the
heart and nervous system — were greatly impaired. Now we know
what an important part the liver plays in the metabolism of carbohydrates,
so that we can easily understand that under the above conditions the
function of the organ is of especial importance to the animal.

How economical the animal organism is with its materials that it has
once built up, is shown by another of E. Pfli'iger's ^ observations. The
larva of the nurse-frog (Ah/tes obstetricans) is fully grown at the end of
May. It has then attained a length of about 8.1 centimeters, of which
about 3 centimeters belong to the real body, and the remainder to its
over-sized tail. After the larva has reached this stage, it no longer takes
any nourishment. At the same time the tail begins to shrivel up. Its cell-
material is liquefied and migrates to the true body; and as the tail dis-
appears, the front and hind legs shoot out. Just imagine what important
transformations must take place in this process of developing the limbs of
the animal from what was the tail! As soon as the tail has all been
absorbed, nourishment is again taken up from the outside.^

This suffices to give us some idea as to the mutual relations in the metab-
olism of the different organs. It is not likely that the above observations
represent exceptional cases. It is far more probable that such changes
are of common occurrence, and in a way this is quite similar to the rela-
tion known to exist between the glycogen in the muscles and that of
the liver.

Even although the greater part of the nourishment absorbed is employed
for the production of energy, a certain portion of it is taken, as required, —
whether albumin, carbohydrate, lecithin, cholesterol or nuclein substances,
— and utilized for the building up and extension of the cells. We cer-
tainly cannot limit our conception of the term " food " to those substances
which we know to be sources of energy. The function of serving the

' E. Pfliiger: Pfluger's Arch. 108, 115 (1905).

' Pfluger's Arch. 29, 78 (1882); 54, 33.3 and 403 (1893).

' The phenomenon is not pecuUar to the larvae of the nurse-frog, but is common to
larvae of amphibia which pass through this stage. The larvae of Rana fusca and of
Rana temporaria at least show a similar behavior, although here the animal apparently
eats up the tail. The material of the organ is utilized, at all events.

354 LECTURE XVI.

cells must also be included. As we have said, water and inorganic salts,
neither of which imparts to the organism any chemical energy, must neces-
sarily be considered as foods, for they exercise this function of being of
use to the cells. Now we know that the animal organism takes up a
considerable amount of salts with its food, while on the other hand it is
equally well known that the organism constantly eliminates salt in the
urine and in the sweat. These losses must naturally be replaced. For
the salts it would seem, a priori, as if there were not much need for such
replacement. We could easily imagine that the salts set free in the break-
ing down of cell-material could be used anew in the formation of new cells.
With water it is quite another matter, because the organism needs water
for a number of different processes. Its importance is shown by the fact
that two-thirds of the animal organism consists of water. Every cell
must contain water. It forms one of the prime conditions for a definite
physical consistency of the cell. Water is absolutely necessary as a
solvent for numerous compounds. It brings into play numerous chemical
reactions, and takes part in the building up and breaking down of sub-
stances without number. It is a carrier of nourishment to the body,
whether through the blood, the lymph, or the finest fissures between the
cell; and, conversely, it provides the means for carrying away the waste
products. When we remember in addition that the body must give up
water to the air in the process of respiration, and that in water the animal
organism possesses its most important means of regulating the temperature
of the body, by virtue of its evaporation on the surface, it soon becomes
apparent why water plays such an all-important part in the life process
not only for animals but naturally for plants as well. In the combustion
of the food, naturally some water is formed in the body, but this amount
is so small that it by no means suffices to satisfy all the requirements.
The animal organisnt must have a supply of water from without.

Now, are the inorganic salts also indispensable for the nourishment of
the fully developed organism? An attempt has been made to answer
this question by providing animals with food which is as free from ash as
possible. The fir.st to perform such experiments was Forster.' As food
he made use of the meat residue obtained in the preparation of Liebig's
Extract of Beef. After having been repeatedly boiled with water, such
meat contains only 0.8 gram of ash, for each 100 grams of dry substance.
This, together with fat, sugar, and starch, he fed to two dogs. Both of
the animals experimented upon died very soon; in fact, much more quickly
than if they had not been fed at all. The same result was obtained by
feeding three pigeons with starch and casein. Against the conclusion
that death was caused by lack of salt, G. von Bunge ^ very properly raised

' Z. Biol. 9, 297 and ,369 (1873).
' Ibid. 10, 111 and 1.30 (1874).

INORGANIC FOODS. 355

the question that possibly the lack of salt may have had an indirect action.
In the catabolism of that part of albumin containing sulphur, the cystine,
there is formed, as we have seen, a considerable amount of sulphuric acid.
This under normal conditions will unite with the ba.sic salts contained in
the nourishment and be eliminated as a salt of the acid. If now the
nourishment contains none of these basic salts, then the sulphuric acid
will constantly withdraw alkali from the cell components so that the
system will not only fail to have salts, but the whole structure of the cells
will be injured by taking away a part of the building material. Now if
such a hypothesis be correct, then the addition to the nourishment, which
is otherwise practically free from ash, of sufficient alkali to unite with this
sulphuric acid should enable the animal to live longer. Lunin ' showed
by experiments that this was actually the ease. He fed mice with casein,
fat, and cane-sugar. The amount of ash contained in this mixture was
only one-tenth of that in the mixture used by Forster. With this food
and distilled water five mice lived respectively, 11, 13, 14, 15, and 21 days.
Other mice were not given any food at all; two died in three, and two in
four days.

Next Lunin fed six mice with the same mixture, to which some sodium
carbonate had been added. These animals lived 16, 23, 24, 27, and 30
days, or nearly twice as long as the mice did in the previous experiment.
Now the objection may be raised to this last experiment that here the
sodium carbonate may not act, as Bunge reasoned a priori, as an alkali,
but rather as a salt. In order to meet this objection, Lunin fed seven
mice with the same mixture, except that the sodium carbonate was replaced
by an equivalent amount of sodium chloride (common salt). These
animals died at the end of 6, 10, 11, 15, 17, and 20 days; i.e., they
did not live any longer than the mice in the first experiment. The experi-
ments were repeated with potassium carbonate and potassium chloride,
but with the same result.

Now although the addition of sodium and potassium carbonate to such
a diet was sufficient to prolong the life of the animals, it was not able to
maintain their existence for any length of time.- Note, however, that
these animals had received only one salt in the nourishment. It might
be thought that better results would be obtained with a mixture of salts.
Lunin, therefore, compared the lengths of life of mice fed upon cow's milk,
with that of mice fed with the above mixture of casein, fat, and cane-
sugar, plus the same salts that are contained in milk. Care was taken to

' Ueber die Bedeutung d. anorganischen Salze f. d. Ernahrung d. Tieres. Dissert.
Dorpat, 1880, and Z. physiol. Chem. 5, 31 (1881).

' Cf. C. A. Soein, Z. physiol. Chem. 15, 93 (1891). Abderhalden u. Rona, ibid. 42,
528 (1904). Henriques and Hansen, ibid. 43, 417 (1905). Falta and Noeggerath, Hof-
meister's Beitrage, 7, 313 (1905).

356 LECTURE XVI.

maintain the same proportion of salts to organic material as in milk. Fed
with such a mixture, six mice lived 20, 2.3, 29, 30, and 31 days, or about
the same length of time as the mice fed with the former mixture containing
alkali carbonate. Two mice, fed entirely upon cow's milk for a period of
2i months, remained in good health at the end of the experiment.

These experiments apparently prove that it is not possible to keep mice
alive without feeding them salts, and, moreover, that an artificial mixture
of salts fails to sustain the lives of mice for more than a short time. This
result may be accounted for in a number of different ways. It has not
shown that the early death of the mice in the first experiment was actually
due to a lack of salts in the diet. It is equally conceivable that certain
necessary organic constituents were wanting. Milk always contains,
besides casein, a certain amount of albumin. It is possible that the
albumin is of great importance for certain functions. Again, perhaps
lecithin and cholesterol are e.ssential. Possibly milk contains organic
compounds of a nature unknown to us. Above all we must remember
that the exceedingly important Law of the Minimum ^ holds for the nour-
ishment of animals as well as for that of plants. In any diet the amount
of each constituent required by the organism must be regulated in accord-
ance with this principle. Perhaps some inorganic element, such as fluor-
ine was lacking, and for this reason the other inorganic constituents were
not sufficiently utilized. Naturally the same law holds with regard to the
organic constituents.

Until recently but little was known concerning the physiological impor-
tance of salts in the plant and animal organism. It was known that they
took part in the anabolism of the cells. In fact, potassium, sodium, calcium,
magnesium, iron, phosphoric acid, fluorine, and chlorine are invariably
found in every cell. In some cases, manganese, silicic acid, iodine, and
arsenic are found in the animal cells. The plants receive their nourish-
ment from the ground, and under certain conditions may contain other
elements; for example, copper, zinc, and aluminium. Silicic acid in many
cases of plant life is an important source of rigidity. The plant cell
requires inorganic material. There is no doubt that the physiological
importance of the salts is the same in both the animal and vegetable king-
doms, and the assumption that they form merely a passive building
material for the cells is not justifiable. This is evidenced by the fact that
the distribution of the inorganic material is not uniform throughout the
organism. Thus we find in certain cells more potassium and less sodium,
while in the liquids of the body, for example the serum, the relative
amounts of the two are reversed. In the cells there is more phosphoric
acid, but less chlorine. It is evident from this that the taking up of salts

' J. Liebig: Die Chemie in ihrer Anwendung auf Agrikultur und Physiologie,
p. 332 (1876).

INORGANIC FOODS. 357

by the cells is an active process. Thus the cell withdraws from the serum,
which is rich in sodium salts and contains le.ss potassium, the potassium.
It is possible that the researches of Bokorny ' concerning the behavior of
lower organisms to solutions of certain dyestuffs may throw some light
on these processes.

He showed, namely, that the protoplasm of certain cells would take up
definite compounds even from very dilute solutions, so that, for example,
a colored solution would eventually become colorless. Bokorny assumed
that chemical combination took place between the protoplasm and the
substances in question. He showed, for example, that the Spirogyra and
Cladophora would absorb silver from a solution containing only one part
in 100,000,000. In a solution of 1 : 10,000,000 enough silver was taken up
so that when treated with hydrochloric acid and hydrogen sulphide the
algsB turned black. Likewise from very dilute solutions of copper and
mercury salts the cells would remove the inorganic material. Toward
other compounds, e.g. gold salts, the behavior was quite different. From
gold solutions containing one part in 100,000 the gold is not removed by
the above-mentioned algae, nor by yeast cells. There is a specific action
between these cells and the heavy metals which we cannot explain at
present. The absorption of certain salts by the cells of the animal organism
must take place in conjunction with certain definite and specific processes
by means of which a definite selection in definite proportions is made
possible. Quite recently we have become able to explain in a measure
the part played by salts in the life of the cells. The function of the salts
is undoubtedly chiefly an osmotic one. It is the task of the inorganic
salts to regulate the osmotic pressure of the cell-fluid itself and of the
intercellular fluid. It is clear that any change taking place in this pres-
sure, whether by the taking up or giving off of water, causing a swelling
or a shrinking of the cell, or, on the other hand, any changing in the
concentration or dilution of the reacting mixture contained in the proto-
plasma, will immediately lead to most serious disturbances in the
progress of certain reactions. Above all, the velocity of the reactions will
be changed.

The part played by the inorganic material is not restricted, however, to
maintaining this constant osmotic pressure between the liquids within
and without the cell. This is evident from the fact that the cell requires
certain definite salts. It is not possible, for instance, to replace success-
fully the potassium in the cell by an equivalent amount of sodium. The
individual salts must exert for themselves a specific action, although it is
not yet clear as to just what this may be. We are acquainted with quite
a number of isolated facts in this connection, but with our present knowl-
edge it is not possible to group them together and view them from one

' Chem.-Ztg. 29, 1201 (1905).

358 LECTURE XVI.

standpoint so that definite conclusions may be drawn. It may be well to
cite one or two examples showing that, in fact, there is a specific action of
the inorganic salts. Overton ' has shown that the muscles of a frog will
retain their normal volume in a 0.7 per cent solution of common salt, and
remain excitable at the end of 40 to 48 hours. In concentrated salt
solutions their volume is diminished, whereas in dilute solutions it increases.
Solutions of grape-sugar, cane-sugar, milk-sugar, mannitol, alanine,
asparagine, etc., having the same osmotic pressure as the 0.7 per cent
common salt solution, are equally indifferent as regards the osmosis. In
such solutions, however, the excitability of the muscle is soon lost. On
adding a little common salt to one of these solutions, however, it is again
possible to excite the muscle. In fact, 0.068 to 0.074 per cent of salt suffices
to render this effect. The next question is whether a change of the anion
(CI) keeping the cation (Na) constant will have any effect. It was found
the chloride could be replaced successfully by equivalent amounts of the
bromide, nitrate, sulphate, bicarbonate, chlorate, acetate, and secondary
phosphate of sodium, showing that the anions had no influence here. In
a series of further experiments the cation was changed, and it was found
that the sodium could be replaced by lithium alone, while potassium,
calcium, magnesium, strontium, and barium salts were unable to preserve
the excitability of the muscle. It is perfectly obvious, therefore, that the
sodium ions, besides serving to maintain a definite osmotic pressure, also
exert a quite specific action upon the contractility of the muscle.

Jacques Loeb ^ succeeded in performing a very pretty experiment. If
the medusa Gonionemus be placed in a solution of cane-sugar or of glycerol,
the osmotic pressure of which corresponds to that of the ocean, its rhythmic
pulsation ceases immediately. This is not the case, however, if a solution
of sodium chloride or bromide is used in the above experiment.

Loeb showed, moreover, that the presence of the sodium ions alone
was not sufficient to maintain the contractility of the muscle. In a 0.7 per
cent solution of common salt the muscles of a frog after about an hour
exhibit rhythmic contractions which last for over 24 hours. It appears
as if the sodium ions irritate in some way the muscular fibers. It has
even been stated that they have a poisonous effect. It is exceedingly
interesting that it is possible to combat this irritation of the sodium ions
(also obtained with rubidium and caesium ions) by the addition of the
bivalent calcium, strontium, magnesium and manganese ions. This
effect is not, however, due merely to the valence of the ion, for the bivalent
barium, zinc, cadmium, and lead ions do not act in the same way. On
the other hand the monovalent potassium ion has an effect opposite to
that of the monovalent sodium ion. It is particularly interesting that

■ Pfliigei's Arch. 92, 115 and .346 (1902).
' Am. J. Physiol. 3, 383 (1900).

INORGANIC FOODS. 359

such closely related chemical elements as sodium and potassium should act
so differently physiologically.

A quite similar observation was made with the Fundulus heleroclilus}
This little fish is not at all sensitive to variations in osmotic pressure.
It exists in salt water as well as in distilled water, while on the contrary,
when placed in a solution of pure sodium chloride of the same concentra-
tion as the ocean, it soon dies. Its eggs behave similarly. If to the
solution of pure sodium chloride the ions of calcium, barium, strontium,
magnesium, lead, cobalt, ferrous iron, zinc, manganese, chromium or alu-
minium, are added, the injurious effect of the sodium chloride is combated
successfully. On the other hand, the ions of mercury, copper, cadmium,
nickel, and ferric iron are without influence.

The dependence of the cell-function upon the nature of the salts present,
and the antagonistic action of different salts, are shown very well by the
following experiment: A salt solution of the concentration correspond-
ing to sea-water is poisonous to the eggs of the fundulus. Calcium and
magnesium exert no recognizable efTeet upon the eggs. If a fundulus
egg be placed in a pure aqueous solution of either of the two last-men-
tioned salts, it does not develop. Development takes place, however,
immediately on adding a sodium salt to the solution.

At this place, we will recall the experiments of Martin H. Fischer.^ By
the injection of a J molecular solution of common salt into the veins, he
was able to produce a glucosuria which in its entire behavior corresponded
to that produced by the diabetic puncture. Such a glucosuria can, in the
case of rabbits, be prevented Ijy adding a little calcium chloride to the
solution which is injected (975 cubic centimeters of J molecular NaCl solu-
tion + 25 cubic centimeters of f molecular CaCl2 solution). After the
elimination of sugar has ceased, it can be renewed by the injection of pure
sodium chloride solution, and again checked by means of the calcium
chloride solution.

This reciprocal effect is interesting, and its study opens up new fields of
investigation. Each cell has evidently particular salts in specific appor-
tionment. A disturbance of this relation by the preponderance of this
salt at one time, and that salt at another time, may, cause considerable
trouble in the economy of the cell. For the present it is necessary for us
to study the action of the individual ions separately and in artificial mix-
tures. As regards the proportions of the separate ions in the cells, our
present methods of investigation can throw no light. It is true that by
analyzing the ash, we can get some idea as to the inorganic constituents
of an organ. Aside from the fact that such an analj-sis can never give us

' Jacques Loeb: Pfluger's Arch. 80, 229 (1900). Cf. W. A. Osborne: J. Physiol. [Proc.
Physiol. Soc. 33, 10 (1905)].

' Pfluger's Arch. 106, 80 (1901), and 109, 1 (1905).

360 LECTURE XVI.

a conception of the way the different elements are combined in the cell,
we need only to refer to the extremely delicate mechanism in the action
of the individual ions to make one realize how far we are from under-
standing fully the mechanism of the cell itself.

Jacques Loeb by means of his comprehensive studies deserves great
credit for having added so much to our knowledge concerning the action
of salts and their reciprocal action, and especially by his work on arti-
ficial parthenogenesis.' At this place we can hardly take up in detail
these numerous and highly interesting investigations. We shall come
back to them again. A great number of such experiments have been made.
Unfertilized eggs of the Annelida may be developed by placing them
in sea-water to which a small amount of potassium salt has been added
(e.g., one or two cubic centimeters of 2i N.KCl or KNO3 solution to 100
cubic centimeters of salt water.) Such eggs develop apparently normal
larvae. Such a solution has no action upon the unfertilized eggs of the
sea-urchin.

These experiments have been cited to show that the presence of inorganic
ions is indispensable for the life-process. The manner in which they
act is still unknown to us. It is possible that new light may be thrown
upon this question by a study of the other components of the cell, and
especially of their behavior towards the ions. Now the cells contain
colloids, and in fact the life process itself is intimately related to their
presence. There is no doubt that the peculiar physical condition of the
cell-contents is of great importance to all of the different processes which
take place within the cell. Indeed, this alone makes it possible for so
many different reactions to take place side by side. Colloidal solutions
diffuse but very slowly — in fact, scarcely at all — ^into one another. In-
creased viscosity of a medium, however, does not affect the rate of diffu-
sions of crystalloids and electrolytes, nor the mobility of the ions, nor does
it affect the degree of dissociation of electrolytes. A monomolecular reac-
tion (e.g., the catalysis of methyl acetate by dilute hydrochloric acid)
takes place in a jelly just as rapidly as in water. On the other hand, the
colloid, on account of its internal friction, often prevents the formation
of precipitates. This is effected, not by preventing the reaction from
taking place, but rather by keeping the newly formed molecules in such
an extremely minute state of subdivision that they do not come together
to form visible complexes. The colloids cause an enormous increase in
surface tension.

The entire conception of colloids is in a stage of rapid development.
There is no way of defining precisely the part that they take in the life of
the cell. Many isolated facts indicate that the future investigation of

' Cf. Abderhalden : Arch. Rassen-und Gesellschaftsbiologie, Jg. I, p. 656 (1905).

INORGANIC FOODS. 361

colloids will undoubtedly greatly increase our knowledge concerning the
work of the cell.' Here we are especially interested in the behavior of
colloids towards ions. Hardy ^ has shown that ions exert a particular
influence upon the condition of the colloid. Negatively charged colloids
are precipitated by the electropositive cations, and positively charged
colloids by anions. In these relations we find a new proof of how extremely
delicate the whole mechanism of the cell is to enable it to maintain
between tHe individual ions on the one hand, and the colloids on the
other in such a relation that all of its functions can take place unhindered.
It is perfectly clear without further explanation, that owing to the various
reactions taking place within the cells, at one time the action of one ion
is most prominent, while at another time it is that of a different one.
The cell must always be able to neutralize at a given moment the action
of any one ion. Undoubtedly physical chemistry has here pointed out
new paths for further investigation, and there is no question but that it
will enable us eventually to draw new conclusions along lines that have
already been studied. Here again in the operations of the delicate mechan-
ism of the cell, it is not right to attempt to distinguish between the physical
chemistry and physiological chemistry of the cells. Here and there it is
advisable to separate the two fields and allow them to develop individually,
but again and again they must come back to a common basis and
unite to form a broader field, which develops by different methods as
a whole. '

It is obvious from a study of the experiments already cited that salts
and water are fully as important as regards the life of the cell as its organic
nutriment. Just as the part played by the latter is quite a varied one,
so in the same way the inorganic substances participate in a number of
different processes. Although they do not furnish the body with energy,
nevertheless they do come into play during the expenditure of muscular
effort, whether by changes in the concentration of the solutions, or by
variations in the osmotic pressure, or of the surface tension, etc.

Inorganic salts are usually present in the different foods to an extent
entirely sufficient for all our demands. An exception to this general rule
is the fact that man and certain animals require an additional supply of
sodium chloride. Sodium chloride is the only inorganic substance which
it is necessary to add to our diet. This is rather remarkable, because both
animal and vegetable food already contains considerable sodium and
chlorine. The explanation of this exceptional requirement in the case of

' a. HansAron: Biochem. Zentr. 3, 15, IG, 17, pp. 461 and 501 (1905). R. Zsig-
mondy: Zur Erkenntnis der KoUoide, Jena, 1905. H. J. Hamburger: Osmotischer
Druck u. loncnlehre in den medizinischen Wissenschaften, Wiesbaden, 1904. R.
Hober: Physikalische Chemie d. Zelle u. d. Gewebe, Leipsic, 1902.

» Z. physikal. Chem. 33, .385 (1900).

362 LECTURE XVL

common salt we owe to G. von Bunge.' Bunge pointed out in the first
place that among animals only the true herbivora, and never the carnivora,
crave salt. This fact is familiar to hunters. They know that wild her-
bivora — ruminants and solidungulates — frequent the salt licks. Now
the amount of sodium chloride which these animals obtain in their food
is about the same per unit of the animal's weight as that obtained by the
carnivora in its diet. It is hardly to be said, therefore, that there is a
lack of sodium chloride unless we assume that this salt plays a particular
part in the organism of the herbivora, an assumption which, in the light
of recent investigations concerning the action of the individual ions, no
longer seems so improbable. Bunge has shown, however, that vegetable
food differs from animal food by the amount of potash which the former
contains. The herbivora obtain three or four times as much potassium
salt in the food as the carnivora do. All vegetables, especially potatoes,
clover, and meadow hay, contain large amounts of potash. We know of
but very few land plants which, like the varieties of Chenopodium and
Atriplex, contain more sodium chloride than potassium salt. It is easy
to account for the high potash content of plants by the distribution of the
elements sodium and potassium on the earth's surface. By the weathering
of silicate rocks, sodium carbonate is formed, which dissolves readily in
rain water and trickles down into the earth. The potassium, on the other
hand, remains with the other bases combined with silica and aluminium as
an insoluble double ^alt. The latter remains near the earth's surface,
while the sodium is flushed out by springs, brooks, and rivers, and carried
on to the ocean. This accounts for the fact that potassium salts pre-
dominate on the surface of the earth, while the ocean is rich in sodium
salts, especially the chloride.

Now why should an increased amount of potassium salts create a demand
for a greater supply of sodium chloride? Bunge suggests the following
ingenious explanation: If a potassium salt, for e.xample the carbonate,
comes in contact with sodium chloride, a partial decomposition takes
place, a little potassium chloride and sodium carbonate being formed.
Now, as regards the inorganic salts of the blood-serum, sodium chloride
ranks foremost. It is found there to a considerable extent, and, as far as
we know, the amount is kept fairly constant. Now, on bringing the serum
in contact with the abundance of absorbed potassium salts obtained from
the vegetable nourishment, this double decomposition between the sodium
salt in the l^lood and the absorbed potassium salt will take place to some
extent. In this way potassium chloride is formed, and a part of the sodium
of the blood combines with the acid which was previously united with the
potassium. By this process the composition of the blood is changed.

' Z. Biol. 9, 104 (1873); 10, 111, 295 and 323 (1874); Lehrbuch der Physiologic der
Men.schen, Bd. ii, p. 103 (1901).

INORGANIC FOODS. 363

The serum now contains a substance which did not occur in it previously,
or at any rate not to the same extent as now; namely, the newly formed
sodium salt. It is the duty of the kidneys, as we shall see later, to keep
guard over the serum and to regulate its chemical composition. They
eliminate every constituent which under normal conditions is foreign to
it, and take away any excess of compounds which belong there. In the
above case the kidneys eliminate the newly formed sodium salt together
with the potassium salt. This process, therefore, results in the serum
being deprived of sodium chloride.

Bunge succeeded in testing this theory experimentally. He himself took
18 grams of K2O as phosphate and citrate in three doses during the course
of the day, and showed that as a result his body lost 6 grams of sodium
chloride. This does not constitute an abnormal amount of potash salts.
A man fed largely on potatoes will easily take 40 grams of K2O into his
system during the day. The loss of sodium chloride is by no means
restricted to the blood. There is a constant exchange of material between
it and the cells. After what we have seen with regard to the effect of the
ions, we can easily understand the possibility that a diminution in the
amount of sodium contained in the cells, which is in no way replaceable
by potassium ions, may lead to serious disturbances. The organism, at
all events, will attempt as soon as possible to restore the disturbed equili-
brium.

Bunge, to support his views, cited numerous facts. He showed, for
example, that in France the country folk consume three times as much
salt per capita as those who dwell in cities. Now it is a fact that in the
country much larger quantities of vegetables are eaten than in the city,
where the diet consists largely of meat. A further support of the assump-
tion that the vegetables rich in potassium were the cause of the increased
consumption of salt, was gained by a study of people who live almost
entirely upon meat; e.g., certain races of hunters, fishermen, and nomads.
To gain this knowledge, Bunge read through a large number of articles on
travels, and also placed himself in correspondence with travelers. In this
way he established the fact that at all times and in all countries where
the people subsist solely upon animal nourishment, either they have no
knowledge of salt, or do not care for it, whereas in countries in which the
inhabitants subsist mainly on vegetables there is always such an unmis-
takable craving for salt, that it has l^ecome considered as one of the neces-
sities of life. This is the case in both the north and south polar regions.
Again, in countries in which the inhabitants live on meat and rice, there is
no craving for salt. Rice contains but one-sixth as much potash as
wheat, rye, barley, or Indian corn, one-twentieth as much as the legumes,
and only from one-twentieth to one-thirtieth as much as potatoes.

Now what happens in the case of a people subsisting chiefly on vegeta-

364 LECTURE XVI.

bles, and yet living where there is no salt supply? Such people prepare
a salt of their own. Thus, Bunge ' was able to procure a salt obtained by
ignition of a plant which was used by the negroes in the southern part of
Khartum, Africa, as a seasoning for their vegetable food. The analysis
of this salt showed it to contain 19.27 per cent Na20 and 4.92 per cent
K2O, or nearly six equivalents of soda to one of potash. It is interesting
here to find that the natives have selected a plant (Salsola, or salt-wort)
which is especially characterized by its high soda content. The natural
instinct, however, does not always assert itself so well in this direction,
for there are other races which use for salt the ash of a plant rich in potash.
Lapicque ^ examined such a salt. The inhabitants of the Angoni district
in British Central Africa use a substance prepared by burning goat manure
and wood. Analysis showed that it contained 21.98 per cent KCl and
0 . 47 per cent NaCl.^ It is interesting to learn, however, that after Lapicque
had shown the natives how to obtain common salt, they gave up the prepa-
ration of their own native condiment. Salt, for the inhabitants of Angoni,
is an extremely expensive article of commerce. The natives toil for salt
upon the plantations.

The assumption that the high potash content of vegetable foods causes
losses in the sodium content of the blood and indirectly of the tissues, is
not in agreement with certain observations. Thus, Landsteiner * fed a
number of young rabbits exclusively upon meadow hay for .3j months.
At the same time another lot of similar animals was fed entirely with
cow's milk, which contains for one equivalent of soda only 0.7 to 3.7
equivalents of potash. Now although these two series of animals were
fed with nourishment containing quite different relative amounts of alkali,
nevertheless, at the end of the experiment the soda and potash content of
the blood was the same in each case. We know, furthermore, that in
spite of the fact that rabbits and hares live on fodder rich in potash, they
do not show the slightest craving after salt, and under normal conditions
do not obtain any in addition to what their food contains. It is possible
that the organism of these animals may be different in some way, so that
the loss of sodium is avoided. On the other hand, it is a well-known fact
that purely herbivorous animals, such as cows and sheep, can subsist upon
fodder rich in potash for a- long time without any extra salt, and there is
no recognizable disturbance in the development of these animals. It is
indeed possible, and even probable, that the potassium salts contained in
the fodder do not have such a marked effect as pure potassium chloride,
when taken by itself into the system at one time and absorbed as such.

' Z. Biol. 41, 484 (1901).

» L'AnthropoIogie (1896).

3 Abderhalden: Pfliiger's Arch. 97, 103 (1903).

« Z. physiol. Chem. 16, 13 (1892).

INORGANIC FOODS. 365

We perhaps do not yet know all the different ways in which potash can be
eliminated. We shall see that for the heavy metals the intestines are an
important vehicle for their elimination. It is even possible that the liver
regulates the amount of potassium salts, holding a part back so that the
blood does not at any one time come in contact with large amounts of
them. At all events, the influence of potassium salts contained in the
food upon the elimination of sodium salts by the urine must be tested with
some food, such as potatoes, which is rich in pota.ssiura salts. The exper-
iment should extend over a considerable period in order to determine
whether any loss of sodium chloride is permanent or only temporary.

The organism must in every case have ways and means for keeping the
soda and potash content of the blood constant in spite of variations in the
food supply. It is worthy of mention that the serum of all species of
animals ' which have been investigated up to the present time always
contains the same amounts of these two elements; the serum of the car-
nivora, as well as that of the herbivora, contains about 0.43 per cent of
soda, and 0.026 per cent of potash. Perhaps the fact that the red cor-
puscles of the ruminants, in contrast to those of the horse, cow, rabbit, etc.,
contain considerable amounts of soda, may shed some light upon the fact
that the former crave salt, while the latter do not. To be sure, the red
corpuscles of the carnivora also contain larger amounts of soda. It is
very interesting that in the milk of carnivora the two alkalies are present
in approximately equivalent amounts, whereas in the milk of the herbivora
and in human milk the potash predominates. The organism of the her-
bivora and of the carnivora, corresponding to their later nourishment,
thus early becomes accustomed to a definite relation between the amounts
of potassium and sodium. The beasts of prey, which live upon the entire
animal, obtain sodium and potassium in almost equivalent amounts. On
the other hand, the herbivora and the human race receive in many foods
the two bases in the same relative amounts as in milk; some kinds of hay
contain three equivalents of potash to one in soda, while in milk there are
from one to six equivalents of potash to one of soda. We may, indeed,
assume that the organism is adjusted to the general preponderance of
potash over soda, and that disturbances take place only when the cus-
tomary relation is changed greatly at the expense of the sodium, as would,
for example, be the case if the food consisted entirely of potatoes. Rye,
peas, and beans likewise contain very considerable amounts of potassium,
as the following table prepared by Bunge shows:

' Abderhalden: Z. physiol. Chem. 25, 65 (1898).

366

LECTURE XVI.

1 Equivalent Na^.O Corresponds to:

Equivalents
K,0.

Beef-blood

White of hens' eggs
Yolk of hens' eggs . .
Organism of mammals
Milk of carnivora . .

Human milk

Milk of herbivora ....
Beef

0.07

0.7

1.0

0.7 to 1.3

0.8 to 1.6

1 to 4

0.8 to 6

4
12 to 23
14 to 21

Wheat

Barley

Oats . . .
Rice ....
Rve ....
Hay ....
Potatoes .
Peas ....
Strawberries
Clover . . .
Apples . . .
Beans . . .

Equivalents
K.,0.

15 to 21

24

9 to 57

3 to 57

31 to 42

44 to 50

71

99

100

110

Bunge's conception that common salt widens the circle of our food
supply, may well be a correct one. It makes it possible for us to enjoy
potatoes and many other foods which are rich in potash. «

As stated before, our ordinary food contains sufficient quantities of the
inorganic salts, and it is, in general, not to be feared that too little of one
or another salt will be taken into the system.

Our diet is ordinarily a mixed one. If one article of food or another
contains too little of any salt, the deficiency is made up by something
else that is eaten. The fact that an exclusive diet of substances lacking
in this or that salt may lead to disturbances will be shown later. Now,
although it be granted that we need take no thought concerning the supply
of inorganic material required by adults, the question arises whether the
customary food of growing individuals likewise satisfies the requirements
of the organism. This is a justifiable question, particularly in the case of
mammals, and especially human children; for right in the midst of their
growing period a change takes place in their food which compels us to
compare the composition of their first food, the milk, with that which is
eaten subsequently. The amount of inorganic salts contained in milk
must give us some idea as to the requirements of the young. The follow-
ing table gives a summary of the contents of the ash from human milk and
that of certain animals: '

Man . . .

Dog , . .

Pig . . .

Sheep . .

Goat . .

Cow . . .

Horse . .
Guinea pig
Rabbit

100 Parts by Weight of Milk Contain in Grams:

K20

NasO

CI

Fe„03

CaO

MgO

PjO.'i

0.0795

0.0253

0.0468

0.0008

0.0489

0.0065

0.0585

0.1382

0.0779

0.1656

0.0020

0.4545

0.0195

0.5078

0.0945

0.0776

0.0756

0.0040

0.2489

0.0157

0.3078

0.0967

0.0864

0.1297

0.0041

0.2453

0.0148

0.2928

0.1302

0.0617

0.1019

0.0036

0 1974

0.0154

0.2840

0.1776

0.0972

0.1368

0.0021

0.1671

0.0231

0.1911

0.105

0.014

0.031

0.002

0.124

0.013

0.131

0.0754

0.0700

0.0999

0.0013

0.2417

0.0241

0.2880

0.2516

0.1980

0.1355

0.0020

0.8914

0.0552

0.9966

Emil Abderhalden : Z. physiol. Chem. 26, 4.87 and 498 (1S99) ; 27, 408 and 356 (1899).

INORGANIC FOODS.

367

A glance at the above table shows that the composition of milk varies
with different animals. The values also vary in the case of different
animals of the same species, but during the suckling period the variation
is only within narrow limits. We shall find that the amount of ash and
also of the inorganic material bears a certain relation to the rapidity with
which the tissue is formed as shown by the body weight.

It is interesting now to trace the relations between the composition of
the milk (especially that of its ash) and that of the suckling. It is of
course oljvious that such a comparison will serve to give us merely a rough
idea of these relations, for our present methods of analysis are not suffi-
ciently delicate for us to attempt to explain the way in which the differ-
ent elements are combined. The analysis of the ash, for example, merely
shows us what inorganic material is present, and gives us absolutely no
conception of the manner in which these elements are combined in the
body. We do not know from this whether the phosphoric acid that we
find was present entirely as calcium phosphate or other phosphates, or
whether it represents in part lecithin. At the same time the knowl-
edge of the constituents of the ash forms a basis for further investiga-
tion, and with the above-mentioned limitations gives us some means for
comparison.

Now it is a striking fact, as the figures below will show, that milk has
such a different composition from the elements out of which it is formed;
namely, the blood, and especially the blood-serum. The cells of the milk-
glands must possess the power of selection. Now, what determines the
composition of the milk? Bunge,' with reference to this question, com-
pared the composition of milk-ash with that of the suckling itself, and
found in the case of dogs the following relations:

100 Parts by Weight of Ash Contain in Grams:

Dog a Few
Hours Old.

Dog's Serum.

K,0 .

Na,0
CaO .
MgO .
Fe^O,

PA-
CI . .

11.14
10.6
29.5
1.8
0.72
39.4
8.4

15.0
8.8

27.2
1.5
0.12

34.2

16.9

3.1

45.6

0.9

0.4

9.4

13.3

35.6

2.4
52.1

2.1

0.5

0

5.9
47.6

Z. physiol. Chem. 13, 399 (1889) and .A.rch. Anat. Physiol. 1886, 539.

368 LECTURE XVI.

With rabbits the following values were obtained:'

1 00 Part s by Weight of Ash Contain in Grams :

Rabbit 14
Days Old.'

Rabbit's Milk.

Rabbit's
Blood.^

Rabbit's
Serum.'

Kfi .
Na^O
CaO .
MgO .
FcOj

PA-

CI . .

10.84
5.96

35.02
2.19
0.23

41.94
4.94

10.06
7.92

35.65
2.20
0.08

39.86
5.42

23.75
31.38
0.81
0.64
6.93
11.11
32.66

3.19
54.72

1.42

0.56

0

2.98
47.83

In the case of the guinea pig we find: *

100 Parts by Weight of Ash Contain:

New-Born Guinea
Pig.

Guinea Pig's
Milk.

K,0

8.22
6.94

32.21
3.09
0.23

42.25
9.1

9 69

Na,0

8.99

CaO

31 1

MgO

Fe,0,

3.1

0 17

P,0,

37.02

CI .

12 84

Camerer and Soldner ' have made a similar comparison between human
milk and the ash of infants:

100 Parts by Weight Contain :

Infant.

Milk.

Infant.

Milk.

KjG

7.8

31.4

FesOa ....

0.8

0.6

NaiiO ....

9.1

11.9

P2O5 ....

38.9

13.5

CaO

36.1

16.4

CI

7.7

20.0

MgO

0.8

2.6

' Abderhalden: Z. physiol. Chem. 26, 498 (1899).

' G, V. Bunge: Z. Biol. 10, 323 (1874).

' Abderhalden: Z. physiol. Chem. 25, 65 (1898).

• Abderhalden: ibid. 27, 356 a899).

« Z. Biol. 40, 526 (1900); 41, 37 (1900); 44, 61 (1903).

INORGANIC FOODS.

369

L. Hugonnenq * obtained quite similiar values :

100 Parts by Weight Contain :

Human
Fcptus.

Human Milk.

Human

Foetus.

Human Milk.

K20

Na^O ....

CaO

MgO

6.20

8.12

40.48

1.51

35.15
10.43
14.79

2.87

FejOa ....
P2O5 ....
CI

0.39
35.28
4.26

0,18
21.30
10.75

Except as regards the composition of the human infant and human
milk, we find by a comparison of the corresponding values that there is a
striking agreement between the ash of the young animal and that of the
milk. In the case of human beings, however, we do not find any such
agreement. Bunge explains this fact by the assumption that the ash
content of milk has not only the task of building up tissue, but also serves
in the preparation of the excreta, especially the urine. The more rapid
the growth of the suckling, the less apparent will be the influence of the
latter function. It is, therefore, in general not to be expected that the
percentage composition of the milk and that of the infant will agree so
closely in the case of human beings a.s with animals, such as dogs, rabbits,
and guinea pigs, which require the mother's milk for but a short time after
birth, but are soon placed upon a diet of green fodder. It Ls easy to see
that a milk corresponding closely to the chemical composition of the young
as regards the inorganic constituents will be more suitable for animals
which develop very rapidly, whereas with species which develop more
slowly, the building up of the separate tissues does not take place so uni-
formly and there are not so many changes taking place at the time when
the growing organism changes to another source of nourishment.

We now come back to our first question: Does the suckling when it
abandons the mother's milk and passes to other food receive a sufficient
supply of inorganic salts? The following table gives a summary of the
amounts of inorganic substances contained in the more important foods: ^

Honey . .
Beef ....
Rye ....
Wheat . . .
Potatoes . .
White of egg
Pear ....
Human milk
Yolk of eggs
Cow's milk ,

100 Parts by Weight of Dry Substance Contain :

K2O NajO

0.80
1.66
0.61
0.62
2.28
1.45
1.13
0.68
0.27
1.67

0.0

0.32

0.01

0.06

0.11

1.45

0.03

0.17

0.17

1.05

CaO

0.007
0.029
0.062
0.065
0.100
0.130
0.137
0.243
0.380
1.511

MgO

0.04
0.15
0.22
0.24
0.19
0.13
0 22
0.05
0.06
0.20

Fe203

0.002
0.024
0.007
0.008
0.009
0.000
0.009
0.004
0.024
0.003

P2O5

0.09
1.83
1.03
0.94
0.64
0.20
0.99
0.35
1.90
1.86

0.05

0.28
0.03

0 13
1.32

0.32

0.35
1.60

• Cotnpt. rend. 128, 1419 (1899). Of. Cornelia de Lange: Z. Biol. 40, 526 (1900).
J Bunge: Z. Biol. 45, 532 (1902).

370

LECTURE XVI.

It is evident from these values that most of the foods are deficient in lime
alone, as compared with milk. Now while we are not justified in assuming
that the lime-content of milk is to be considered as the normal amount
required for man's later development, still, on the other hand, we must
not forget that probably the " law of the minimum " holds for animal
organisms as well as for plants. Now lime plays a quite particular part
in the development of the organism, especially for certain tissues, the
bones and teeth. It is clear, therefore, that the system must have an
adequate supply of the lime at its disposal. Of course the fully developed
organism requires lime as well, for even then, as we have already seen,
there is a constant building up and wearing down of tissue, and especially
of bony tissue. The following table gives the amount of lime present in
many of our foods. At the same time the amounts of iron they contain
are also given. The values refer to 100 grams of substance dried at 120° C.
They are arranged with increasing lime-content.

In Milligrams:

In Milligrams:

CaO

Fe

CaO

Fe

Sugar

Honey

Beef

Pig's blood ....
White bread . . .
Malaga grapes . .

Wheat

Rye

Apples

Graham bread . .
Pears

0
7

29

33

46

60

65
62 to 71

66

77

95.
100
103
108
123
126

0

1.2

16.9

225.7

1.5

5.6

5.5
3.7 to4.9

1.9

5.6

2.0

6.4
1.0to2.0

2.1

1.9

2.5

White of egg . .
Red cherries . .

Peas

French plums . .

Plums

Huckleberries . .
Human milk . .
Yolk of egg . . .
Figs ......

Wild raspberries.
Oranges ....

Cabbages (light
green leaves) .
Wild strawberries
Cow's milk . . .

130
136
137
154
166
196
243
380
400
404
575

717
873
1510

0

1.2
6.4
1.8
2.8
6.4
2.3 toS.l
10 to 24
4.0
3.7
1.5

Rice

Bates .....
Black cherries . .
Cocoa beans ....

5.6

8.1 to9.3

2.3

These values show that it is by no means immaterial what food the
infant receives at the time it leaves the mother's breast. When the child
is six months old, it takes about one liter of milk daily. This amount
contains, in round numbers, about 0.5 gram of lime. We do not know
exactly how much lime the nursing infant requires at the time it is
weaned. We can safely assume, however, that in the later periods of its
growth, it requires relatively less. For one thing, the development takes
place more gradually than is the case shortly after birth; and, again, the
absolute amount of lime taken into the system increases with the quantity
of food eaten. The following figures will give some idea of the rapidity of
the development shortly after birth. Rabbits double their weight at the

INORGANIC FOODS. 371

end of six or seven days.' It takes nine days for dogs to accomplisli
the same result; at the end of eighteen days the weight was three times
that at birth. With cats it requires nine or ten days for the weight
to become doubled; in one case the weight was three times as much
at the end of 19^ days, and four times as much at the end of 29i daj's.
Pigs develop less rapidly. On an average, it requires 14 days for
the original weight to be doubled; with sheep 15 days, goats 32 days,
calves 47 days, and 60 days in the case of a colt. The slowest devel-
opment is shown in the case of the human offspring, which does not double
its weight until about six months have passed away. Observations upon
the cat show that the rate of development decreases with age. It is
particularly striking only at the time of birth. The case of guinea pigs is
not without interest. According to their development, they scarcely
belong in the ranks of the mammalia; by eating green food shortly after
birth, they rapidly increase in weight. At birth they are already remark-
ably well developed. Even then they are able to eat the same food as
that of the mother, and thrive on cabbage, etc. The female of this animal
possesses onl}' two mammary glands, situated in the gi'oin, and milk plays
but a subordinate part in the nourishment of the new-born guinea pig.
The fact that the first development of these animals takes place quite as
rapidly as in the case of the most closely related animals, leads us to the
assumption that in early times these animals came into the world in a
much more undeveloped condition, and were forced to depend upon milk
for nourishment, like other mammalia.

It has often been suggested that rickets, a quite common children's
disease, is caused by a lack of lime-salts in the nourishment. In fact, this
disease appears most frequently when the mother's milk for some reason
is replaced by some other form of nourishment. There is no doubt that
in considering the value of a food for replacing the mother's milk, too
much stress has been laid upon the amount of fat, proteid, and carbo-
hydrate. Certainly a mistake is being made unless equal attention is paid
to the amount of inorganic substances contained in the nourishment.
According to general experience, it is not possible to replace the mother's
milk satisfactorily by the milk of some other animal. It is necessary to
add something to cow's milk in order that there may not be any substance
present in less than the proper amount. Then again it is particularly
erroneous to judge the value of a food used to replace the mother's milk
by its calorific value. We must never forget that the suckling must build
up its tissue first of all. It is, therefore, bj' no means a matter of indiffer-
ence whether this or that organic substance is relegated to the background,
whether fat or carbohydrate. For the growing suckling, carbohydrates
and fat cannot be considered as equivalent in this sense. They are

' Abderhalden: Z. physiol. Chem. 26, 487 (1899); 27, 408 and 594 (1899).

372 LECTURE XVI.

isodynamic only as regards their combustion value. The law of isody-
namics would undoubtedly hold in the case of sucklings in this direction
were it not for the fact that its body substance is increased so largely
that it is necessary to arrange its diet accor.ding to particular lines. We
must, to be sure, admit that by the transformation of carbohydrates to
fat, and perhaps also by the reverse process, one of these nutrients may
replace another, even in the construction of the cells.

The assumption that rickets is caused by a lack of lime in the nourish-
ment is contrary to numerous observations. Rickets appears sometimes
with a food that is rich in lime, even when the child is being fed upon
mother's milk, though much less frequently than with other food. It
might be thought that there still may be a deficiency in lime, and perhaps
on account of the fact that, owing to a disturbed process of absorption, an
insufficient supply of lime becomes available to the tissues. This brings
us to the question as to the state of the lime when it is absorbed and
assimilated.

We meet here with one of the most remarkable circumstances in
the entire subject of physiological nutrition. Whereas our knowledge
concerning the occurrence of organic nutrients is considerable, we know
very little concerning the way in which the inorganic substances are com-
bined in the food. We do not know whether they are present as inorganic
salts, — in the animal and vegetable tissue, — or whether complicated
organic compounds are at hand which contain these inorganic elements
in a state of more or less firm combination. It is conceivable that lime, for
example, can take part in the construction of tissue only when it is present
in a definite state of combination. Such an assumption was especially
justifiable at the time when it was not recognized that it was possible for
the human organism to accomplish syntheses. Now that we have seen,
however, that the animal cells are capable of accomplishing most com-
plicated syntheses, it becomes more and more probable that they are also
able to make use of inorganic salts in the formation of their tissue.

Although we know very little concerning the way in which lime is con-
tained in the ordinary foods, still, on the other hand, it is to be expected
that an explanation of the way lime is present in milk will throw the most
light upon this question. There are a number of possibilities to consider.
It may be that the lime is in some way combined with the protein in milk,
or that it may be dissolved in it in the form of an inorganic salt. At all
events, the fact that the lime is not present in any firm state of combination
is proved by the following experiment performed by Bunge.' On diluting
cow's milk with water and precipitating the casein by careful addition of
acetic acid in the cold, only a trace of lime is found in the albuminous

Z. Biol. 45, 5.32 (1901).

INORGANIC FOODS. 373

precipitate. By boiling and concentrating the filtrate, the globulin and
albumin of milk are obtained. These proteins likewise contain but little
lime. It is not impossible, but quite probable, that the small amounts of
calcium contained in these precipitates are merely carried down mechani-
cally, without there being any state of combination between the albumin
or globulin and the calcium. The greater part of the lime is found in the
filtrate from the two precipitations. After the removal of the casein and
of the two other protein substances, the addition of oxalic acid at once
causes the formation of a precipitate. The filtrate from this last precipi-
tate of calcium oxalate was evaporated to dryness and ignited with sodium
carbonate in order to remove the calcium from any organic substance
which might not have been precipitable by oxalic acid. This ash, however,
contained merely a trace of calcium. This shows that the calcium, if
originally combined with protein, must be present in some loose salt-like
combination such that even dilute acetic acid suffices to set it free. It
has, however, not been established definitely whether some other organic
substance in the milk ma}' not serve to keep calcium in solution in the
presence of phosphoric acid.'

From these observations of Bunge we may say that it is undoubtedly
true that lime is absorbable and assimilable as such. Against this assump-
tion the objection may be raised that it is possible that the lime in the
intestine first of all enters into combination with some organic substance
and is then ready for absorption, and that the latter process depends
entirely upon the formation of such an organic compound. On the other
hand, it may be said that even in such a case the manner of combination
in the alimentary canal must be a weak one, for it is not to be assumed
that any sort of firm combination is formed. Such a loose form of com-
bination would have the effect of keeping the calcium in solution, but it is
perfectly certain that they have no other effect upon the process of assimi-
lation. We cannot be wrong in assuming that every calcium compound
which can be converted into a soluble condition in the intestine is capable
of being absorbed and assimilated.

We have up to now found no reason for believing that the disease of
rickets is due to a diminished capacity for absorption on the part of the
alimentary canal.' It is far more probable that the cause of the faulty
calcification of the bones is due to a diminished assimilation of lime. It
is highly probable that the cause of this is not to be sought in an unsuitable
condition of the calcium salts that are at the disposal of the tissues. It is

' L. Vaudin, Ann. inst. Pasteur, 8, 502 (1894), has observed that the amount of
citric acid in milk is proportional to the lime content. It is altogether out of the ques-
tion to believe that the lime is simply dissolved by this acid, but possibly citric acid in
conjunction with other organic substances may serve to keep the lime in solution.

' T. G. Rey: Deut. med. Wochschr. 35, 569 (1895).

374 LECTURE XVI.

much more likely that the function of those cells whose duty it is to assimi-
late the required calcium does not exert itself normally. All the facts
known concerning the bones of children who have suffered from this
disease agree best with this conception. Above all there is a striking over-
production on the part of the osteoplastic tissue. This results in the
formation of soft bones deficient in lime, the so-called " osteoid tissue."
At the same time there is an abnormal resorption of the tissue already
formed. We cannot be far wrong, if we attribute the disease of rickets to
a metabolic disturbance on the part of a certain group of cells; and here
again the cells themselves stand in the foreground as oi'gans of assimila-
tion. We may say in this connection that it is not the cells which play
the chief part in the assimilation of lime. The other component, which
we cannot yet sharply formulate according to our present knowledge, and
therefore designate in general liy the term "plasma," is equally important.
We have said nothing concerning the condition of the lime in the cells;
and, in fact, we are not able at present to depict the calcification process
which takes place in the formation of bones. We do not know whether
the osteoidal tissue formed in the disease of rickets is capable of taking
up lime at all, or whether it is able to deposit calcium salts from their solu-
tions. If we recall what was said concerning the mutual relations of the
individual ions, we shall be tempted to attribute the over-production of
osteoidal tissue to some such influence.' We have seen that calcium
chloride, for example, acts antagonistically towards sodium chloride, and
have drawn the conclusion from all such observations that only by means
of the inorganic substances in the cell acting together do we have any
guarantee for the normal exercise of the functions of the cell. If any one
element is missing, a disturbance must necessarily follow. A certain
amount of opposing force is lost, and thus the action of a certain ion or a
group of ions exerting a similar effect may be felt. With these suggestions
we will merely state that all our present knowledge concerning rickets
leads us to the conclusion that the disease cannot be attributed to a lack
of lime in the diet of the child, and to-day we have no reason for assuming
that the lime is present in the cells of those who are suffering from this
disease in a form which the cells cannot assimilate. The cause of rickets
is doubtless much more deeply seated, and is to be traced to the organiza-
tion and metabolism of the cells and tissues concerned in the process of
bone production. The whole course of this disease, which in fact is usually
" outgrown," is in accordance with this view. It is not to be assumed

' Cf. Clowes and Frisbe, Am. J. Physiol. 14, 17.3 (1905), who have found that a
rapidly developing adeno carcinoma in mice contains considerable potash and less lime.
Slow development of the tumor shows the reverse proportions. It is not possible to
draw definite conclusions at present as to which process is primary and which secondary
in nature, but studies in this direction certainly warrant attention.

INORGANIC FOODS. 375

that more lime is received in the later years, for, as a matter of fact, milk,
which is invariably the basis of infant diet, contains more lime than almost
any other article of food, so that the infant receives relatively more lime
than at any subsequent time.

It is to be expected that the formation of the bones will be seriously
affected if the lime in the food is intentionally made inadequate. Thus
when Forster ' and likewise Voit ^ fed young dogs with meat, fat, and
water, free from calcium, a faulty bone-formation was soon apparent.
Chossat,' and later on Voit/ observed that fully developed pigeons, which
were made to subsist for a year exclusively upon washed wheat grains
and distilled water, showed a deficient skeleton. The bones were very
fragile, the skull and breast-bone being very thin, and penetrated with
sieve-like perforations. These experiments merely prove that the bones
require lime for their development. Such experiments have no connection
at all with the disease of rickets. Particularly, the observations made
with the pigeons remind one very much of osteoporosis, a morbid absorp-
tion of bone which often takes place in elderly people, especially in the
region of the skull, and is obviously due to inadequate nourishment of the
bony tissue.

Up to this point we have only traced the metabolism of the animal
organism in two periods, namely, during growth and after the organism
is fully developed. An especial observation with regard to the content
of the food in inorganic salts is furnished by the organism of the mother
during pregnancy and during lactation. At this time the organism of
the child develops exclusively at the, expense of the mother. If the material
received by the mother in the food during this period is not sufficient,
then the stores of her organism are attacked, and finally her own tissue
is subject to resorption. The foetus develops continually even when the
mother is starving. Remarkable migrations of substance must take
place during this entire process. Our knowledge concerning these rela-
tions is still very incomplete. The following table prepared by Hugon-
nenq ^ gives an idea of the amount of inorganic material taken up by the
foetus during its development.

We see from these values that towards the end of pregnancy there is
suddenly a marked increase in the amount of inorganic material taken up
by the foetus. During the last three months it takes up almost twice as
much inorganic material as during the first six months of its development.
Altogether it withdraws about 100 grams of ash constituents from the

' Z. Biol. 9, 369 (1S73); 12, 464 (1876).

= Ibid. 16, 85 (1880).

= Compt. rend. 14, 451 (1842).

* Ber. Vers. Deut. Naturforscher, Munchen, 1877, 243.

' Compt. rend. 128, 1054 (1899).

376

LECTURE XVI.

mother. Of iron it takes up 0.294 gram (0.42 gram Fe203). Here
again the greater part is taken up during the latter part of the period of
pregnancy. This gives one the impr&ssion that the fcetus is probably
provided with reserves in order to fit it for all contingencies which may
arise with regard to its nourishment after the birth.

Age of the Foetus.

Sex.

Weight

Fe^Oj

of the Foetus
in Ivilograms.

of the Ash

in grams.

of the Entire
Organism.

Calculated to
lOOgrams Ash.

4J months

5 months

5 months

5 to 5i months . . .
5J months

6 months

Towards end of preg-
nancy

Towards end of preg-
nancy

9
9
9
9
9
9

0.52

0 57
0.80
0.12
1.29

1 17

2.72
3.30

14.00

18.72
18.36
28.07
32.98
30.77

96.76

106.16

0.06
0 06
0.07
0.11
0.13
0.12

0.38

0.42

0.43
0.33
0.40
0.38
0.38
.0.39

0.40

0.40

After its birth the child still receives nourishment at the expense of the
mother. Milk now affords the vehicle for the transference of material.
An idea of the amounts of material which the mother has to furnish the
child is shown by the following figures: A male nursling takes about
one liter of milk per day at the age of six months. This contains the
following amounts of separate constituents: ' Water 875.8 grams, casein
8.0 grams, albumin 12.1 grams, fat 37.4 grams, milk-sugar 63.7 grams,
ash 3.0 grams. The ash is composed of 1.08 grams potash, 0.28 gram
soda, 0.50 gram lime, 0.07 gram magnesia, 0.007 gram ferric oxide,
0.66 gram phosphoric acid, 0.53 gram chlorine.

This increased output is naturally felt by the organism of the mother,
and it must be taken into account in the choice of her food. Here again
it would be altogether wrong, as regards the nourishing of the nursling, to
regulate the diet of the mother with regard to the calorific value of the
food. It is the chemical composition of the food which is of utmost
importance, for the milk must provide the infant not only with combust-
ible matter, but above all with building material for its cells. Although
the organism of the child can probably utilize carbohydrates for the pro-
duction of fat, and can perhaps form sugar from albumin, it is impossible
to effect such transformations in the case of inorganic salts. Even the
assumption that the place of one salt may be taken by another one which

' Averages from 173 analyses.
Genussmittel, Berlin, 1904.

See J. Koenig: Die menschlicher Nahrungs-und

INORGANIC FOODS.

377

is closely related to it, is not justifiable, for we have seen that the ions have
a quite specific action. For this reason it seems as if more attention
should be paid to the inorganic nature of the ash of milk, or of any milk-
substitute, in considering its value as food for the child.

It is not right, however, to lay particular stress upon any one inorganic
salt. At present we are not able to judge the relative merits of the different
salts. As we have seen, alrnost all of the inorganic substances, with the
single exception of lime, are present in sufficient quantities in the ordinary
articles of diet.

One hundred grams of dry substance contain the following amounts of
lime in milligrams: '

Beef

29
46
77
100
137

166

White bread

Graham bread

Potatoes

Peas

Human milk

Yolk of eggs

Strawberries

Cow's milk

243
380
483
1510

This little summary shows that a diet consisting chiefly of meat is not
suitable for the nursing mother. Rich in lime are the yolk of eggs, and
especially cow's milk. We can well imagine, a priori, that the organism
of the mother during the entire period when the child receives its nourish-
ment at her expense will suffer materially if there is lack of lime in her
food, and in such cases she will be obliged to draw upon her own supplies
of lime to furnish the child with the amount that it requires.

Osteomalacia, a disease in which the bones gradually lose their solid
constituents and finally become thin as parchment, soft and flexible,
occasionally occurs during pregnancy. It would seem probable that this
disease bears some relation to the increased requirement of lime-salts on
the part of the organism of the mother. The child develops at the expense
of the mother's tissue. All that we know concerning the disease, however,
is contrary to this assumption. It occurs more frequently in certain
localities.^ Its appearance is not restricted to the period of pregnancy.
It is at such a time, however, that the symptoms are most pronounced, and
usually the disease then progresses more rapidly. The histological
study of osteomalacial bones shows that it is not lime alone that the bones
have lost. It is true that one of the most noticeable changes is the decal-
cification of the individual lamellae; but at the same time there
takes place, with varying intensity, a new formation of osteoidal tissue.
The assumption that this decalcification of the bones probably does not
stand in any direct relation to the development of the foetus is supported

> G. von Bunge: Z. Biol. 41, 155 (1900).

' Cf. L. Gelpke: Die Osteomalakie im Ergolztale, Basel, 1891.

378 LECTURE XVI.

by observations of the metabolism which takes place during the disease.
Thus Goldthwait, Painter, Osgood, and McCrudden ' found that when the
nourishment contained 4.56 grams of lime there was an elimination of 3.859
grams by the urine and of 1.80 grams by the faeces. Thus the organism
lost 1.10 grams of lime in the process. Hand in hand with the decalcifi-
cation, a formation of an organic substance takes place. This substance is
characterized by a high sulphur and a low phosphorus content. Curiously
enough, while the calcium is being carried away from the organism, mag-
nesium is being held back. Considering all these facts together, we are
led to the conclusion that the di-sease is not caused by a giving up of lime
to the organism of the child, but that evidently there is a severe metabolic
disturbance of the bony tissue which naturally must be influenced indi-
rectly by the important transformations which are taking place in the entire
metabolism of the organism of the mother due to the presence of the
new being. But just as in the disease of richitis the lime plays a more or
less passive part, it is indeed highly probable that here again the absence of
lime is not directly responsible for the trouble, but that the loss of lime
takes place secondarily as a result of the disease. The lime is loosened
from its state of combination in the bones, and is eliminated as refuse out
of the system. The primary trouble is a disturbance in the economy of
the bony tissue. It has been frequently suggested that the cause of the
decalcification is due to the appearance of acids. This was deduced from
the fact that lactic acid is found in the urine of those suffering from osteoma-
lacia. The appearance of the lactic acid, however, does not prove anything
in this direction. In fact, cases of the disease are known in which no lactic
acid could be detected in the urine; and we know, furthermore, that the
acid may appear in the urine for quite a number of different reasons with-
out the lime-content of the bones being affected at all. The appearance
of lactic acid in the urine does not indicate where the acid originates.
There is absolutely no foundation for the assumption that the acid in
osteomalacia is formed in the bony tissues and serves to dissolve out the
lime.'

Fehling's ^ observation, that removal of the ovaries serves to check the
disease, has shed a peculiar light upon the nature of the disease. After
this operation, lime is once more retained by the system, and the newly
formed osteoidal tissue calcifies. At present we can merely assume that
the loss of the ovaries brings back the metabolism to normal paths. We

■ Am. J. Physiol. 14, 3S9 (1905).

' Cf. C. Schmidt, Ann. 61, 329 (1847). Moers and Muck, Deut. Arch. kiln. Med.
6, 485 (1869). Nencki and Sieber, J. pr. Chem. 26, 43 (1882). M. Levy, Z. physiol.
Chem. 19, 239 (1894).

' Arch. Gynak. 39, 171 (1891); 48, 472 (1895). Cf. also Winckel: Sammlung klini-
scher Vortrage N. F. No. 71.

INORGANIC FOODS. 379

may suspect that the ovaries have previously produced something which
has caused the metaboUc disturbance. Such an hypothesis, however, has
not up to the present time been established experimentally. We must
for the present be content with a knowledge of the observed facts, and
await a fuller explanation of the peculiar mutual action between the
ovaries and the bony tissue as a result of further investigation.

LECTURE XVII.

INORGANIC FOODS.

II.

We have started with milk as a standard for determining the require-
ments of the animal organism as regards inorganic material. This is
justifiable inasmuch as it is certain that milk contains in proper propor-
tions all the inorganic salts which are necessary for the development of
the growing individual. Under ordinary conditions there is little fear of
an insufficient supply of these elements in the case of adults, except,
of course, during periods of pregnancy and of lactation. Our ordinary
mixed diet contains a sufficient amount of all the inorganic substances,
even when on account of social reasons the nourishment is obtained from
material which is not of full value. We shall come back to this point.

In comparing the composition of milk with that of the other foods, we
left one important fact unmentioned; namely, the relatively low iron con-
tent. There are, in fact, but few articles of food which contain less iron
than milk, as is shown by the following table arranged with increasing iron
content : '

Sugar . . .
Egg albumin
Honey . . .
Red cherries
Rice ....
Scotch barley
Oranges . .
White bread
Wheat flour .
Greengage
Black cherries
Apples . . .
Pears ....
Dates ....
Cow's milk .
Human milk
Cocoa beans

Miiligrams of Iron

per 100 Grams Dry

Substance.

0 0
0 0
12
12
1.0-2
1.4-1
15
15
16
1.8
1
1

9

9
2 0
2 1
2 3
3-3.1
2.5

Plums

Raspberries

Figs

Shelled hazel nuts . .

Rye

Cabbage (etiolated

leaves)

Barley

Shelled almonds . .

Wheat

Malaga grapes . . .
Cabbage (light-green

leaves)

Huckleberries ...

Potatoes

Peas

Beans (white) ....

Milligrams of Iron

per 100 Grams Dry

Substance.

2.8
.7-3.9
.7-4.0

4 3
.7-4.9

4.5
4.5
4.9
5.5

5 6
.7-6.

6 4
.2-6.

8.3

' G. von Bunge; Z. Biol. 45, 532 (1901). Cf. Hauserraann: Z. Physiol. Chem. 23,
555 (1897)

380

INORGANIC FOODS.

381

Carrots

Wild strawberries . .

Wheat-bran

Lentils

Almonds (brown skins)
Hazel nuts (brown

skins)

Dandelion greens . .
Beef

Milligrams of Iron

per 100 Grams Dry

Substance.

9.5
9.5

12.7
14.3
16.9

Asparagus

Yolk of eggs . . . .

Cabbage (outer dark-
green leaves of the
head)

Spinach

Pig's blood

Hematogen

Hemoglobin . . . .

Milligrams of Iron

per 100 Grams Dry

Substance.

20.0
10-24

17-38

33-39

226

290

340

The remarkably small amount in milk of an inorganic element to which
we are accustomed to ascribe so great significance is very striking. Iron,
we know, forms an important constituent of the hemoglobin. Now in
comparing the composition of the ash of the suckling with that of the
milk, we found that the former contained considerably more iron. This
obliges us to draw the conclusion that the new-born animal is already
provided with a store of iron. The organism of the female can supply its
young with nourishment in two ways, — at first through the placenta, and
later by way of the mammary glands. Evidently for some reason or other
in the case of iron the former method is preferred. In corroboration of this
Bunge ' showed that the amount of iron contained in a new-born rabbit is
greatest at the time of birth, and decreases from day to day until it reaches
a minimum at the end of the period of lactation, increasing immediately
as soon as the animal changes to a food richer in iron. The following
table shows the results obtained by Bunge:

Embryo arranged ac-
cording to weight

1 hour after birth

I day after birth .

4 days after birth

5 days after birth

6 days after birth

7 days after birth

II days after birth

Milligrams of Iron

per 100 Grams of

Body Weight.

6 4
8.5
9.0
18 2
13.9
9,9
7.8
8.5
6 0
4.3

Ape of Rabbit.

13 days
17 days
22 days
24 days
27 days
35 days
41 days
46 days
74 days

after b
after b
after b
after b
after b
after b
after b
after b
after b

rth
rth
rth
rth
rth
rth
rth
rth
rth

Milligrams of Iron

per 100 Grams of

Body Weight.

4.5
4.3
4.3
3.2
3.4
4.5
4.2
4.1
4.6

Rabbits are fed on the milk of the mother for about three weeks. These
values show that the lowest iron content coincides with the end of the

Z. physiol. Chem. 16, 17.3 (1892); 17, 6.3 (1893).

382

LECTURE XVII.

period of lactation. If the assumption be correct that the organism of
the mother ordinarily provides her young with sufficient iron before the
birth, and that the percentage of iron diminishes during lactation, it
would seem probable that in the case of guinea pigs there would be no
such maximum iron content at the time of birth, for there is no need of
such a supply, inasmuch as the animal immediately after birth begins to
feed on green fodder, which is rich in iron. In fact, as Bunge has shown,
this assumption is verified by the facts:

Age of the Animal.

Milligrams of Iron

per 100 Grams Body

Weight.

Age of the Animal.

Milligrams of Iron

per 100 Grams Body

Weight.

Embryo

6 hours after birth .
li days after birth .
3 days after birth

f4.6
4.4
5.6
15.3
5.0
6.0
5.4

5 days after birth .

9 days after birth .
15 days after birth .
22 days after birth .
25 days after birth .
53 days after birth .

5.7
4.4
4.4
4.4
4.5
5.2

Here, as we expected, there is no maximum of the iron content at birth
as compared with later periods; there is likewise no minimum.

Why does the animal organism prefer, as a rule, to supply the new being
with such a store of iron at birth as wLU permit it to be satisfied with a low
iron content in the milk? Bunge's idea is that the assimilation of iron is a
difficult process. The organism of the mother is, therefore, as economical
as possible with its stores of iron, and prefers that the young should receive
it through the more certain path of the placenta than through the alimen-
tary canal of the offspring.

The importance of the iron stored in the organism may, however, be in
another direction. First of all it is to be decided in what form the iron is
deposited in the new-born animal. We can, indeed, imagine that to some
extent at least it is present as an antecedent of hemoglobin, so that it can
be quickly changed into the latter as occasion demands. On the other
hand, it is also possible that the offspring is provided with a large amount
of hemoglobin itself. It is true that the naked, helpless being which de-
velops so rapidly after birth requires a large amount of oxygen to effect
the various oxidation processes which take place within it, and in order
to make use of all this oxygen it requires hemoglobin. The following
summary shows that, as a matter of fact, the amount of hemoglobin present
per kilogram of the body weight is greatest at the time of birth.'

' Emil Abderhalden: Z. physiol. Chem. 34, 500 (1902).

INORGANIC FOODS.
RABBITS, SERIES I.

383

Age in Days.

Absolute Weight of tlie Hemoglobin

in the Entire Animal Minus the

Intestine.

Hemoglobin per 1000 Grams
Body Weight.

1

0.675
0.699
0.760
0.773
0.981
1.096
1.122

12.61

3

10.91

6

6.61

10

4.87

14

3.81

18

3.21

22

2.41

RABBITS, SERIES II.

Age in Days.

Absolute Weight of the Hemoglobin

in the Entire Animal Minus the

Intestine.

Hemoglobin per 1000 Grams
Body Weight.

1

0.836
0.868
0.999
1.083
1.175
1.384
2.830

13 37

3

11.27

g

6 44

12

5.25

18

4 08

22

3.01

28

5 47

RATS.'

Age in Days.

Absolute Weight of the Hemoglobin

in the Entire Animal Minus the

Intestine.

Hemoglobin per 1000 Grams
Body Weight.

1

0.026
0.048
0 064
0.105
0.221
0.296

12 96

6 •.

6 42

11

4 88

22

28

4.64
6 70

32

7 39

These values which in the case of the rabbits were obtained with animals
of one and the same litter, whereas in the case of the rats a whole litter was
analyzed at the end of each period, show that the absolute amount of
hemoglobin increases slowly after birth. At the end of the lactation
period there was twice as much hemoglobin in the rabbits as at birth. In
the case of the rats, an examination of the contents of the stomach and

' The values for each day were those of a whole litter.

384

LECTURE XVII.

intestines showed that they began to take nourishment, other than the
milk of the mother, at the end of the twenty-second day. During this
time the absolute amount of hemoglobin had increased more than three-
fold. It was perfectly possible for this increase to arise from the amount
of iron contained in the milk. Now if we compare the amounts of hemo-
globin present per kilogram of the animal's weight, it is evident that at
birth the relative amount of hemoglobin present was remarkably high;
this value diminishes little by little as the animal gains in weight, reach-
ing its lowest value toward the end of lactation. This minimum becomes
all the more remarkable when we remember that with full-grown rabbits
there is from 7 to 10 grams of hemoglobin per kilogram of the animal's
weight. As soon as the animal's nourishment changes from milk to
green fodder rich in iron, both the absolute and relative amounts of hemo-
globin increase quite materially.

It is interesting to know how much iron is present in the new-born
rabbits in some other form than hemoglobin. In the following table this
is computed on the assumption that the hemoglobin of rabbits contains
0.336 per cent of iron.' The value thus obtained is then deducted from
the amount of iron that Bunge - found per kilogram of the animal's
weight.'

RABBITS, SERIES I.

Absolute Weight

Hemoglobin

per 1000
Grams Body

Weight.

Milligrams

Milligrams Total

Milligrams Iron

Age in
Days.

of tlie Hemoglo-

Iron as Hemo-

Iron per 1000

not Present as

bin in the Entire

globin per

Grams Body

Hemoglobin per

Animal Minus

1000 Grams

Weight.

100 Grams Body

the Intestine.

Body Weight.

(After Bunge).

Weight.

1

0.675

12.61

42

139

97

3
4

0.699

10.91

37

[99

J62

6

0.760

6 61

22

85

63

10
11

0.773

4.87

16

{43

1-27

13
14

0^981

3.81

13

|45

[32

17
18

l!696

3.21

11

[43

{ 32

22

1.122

2.41

8

43

35

' O. Zinoffsky: Z. physiol. Chem. 10, 32 (1885).

' Loc. cit.

^ This coniputation is naturally not accurate, but gives relative values.

INORGANIC FOODS.

385

RABBITS, SERIES

[I.

Age in
Days.

Absolute Weight

of the Hemo-
globin in the En-
tire Animal Minus

Hemoglobin
per 1000
Grams Body

Weight.

Milligrams
Iron as Hemo-
globin per
1000 Grams

Milligrams Total
Iron per 1000
Grams Body

Weight.

Milligrams Iron

not Present as

Hemoglobin per

1000 Grams

the Intestine.

Body Weight.

Body Weight.

1

0.836

13.37

45

139

94

3
4

0.868

11.27

38

}99

[61

7
8

0^999

6. 44

22

1 60

|38

11
12

l!b83

5.25

'l8

{43

}25

17

18

i;i75

4.08

14

{43

[29

22

1.384

3.01

10

43

33

27
28

2 '830

5.47

18

[34

[..

A glance at these tables shows distinctly that shortly after birth a con-
siderable amount of the iron is present in some other form than hemo-
globin. This amount of iron diminishes rapidly during the first few
days after birth, and is evidently transformed into hemoglobin. After
about the sixth day from birth, the amount of iron not present as hemo-
globin, per 1000 grams of the animal's weight, remains fairly constant.
Inasmuch as the animal is constantly gaining in weight during this period,
it is evident that iron must be continuously deposited in the tissues.
This iron comes from the milk. On the other hand, the absolute amount
of hemoglobin likewise increases. At all events, the above values show
the minimum amount of iron which is in the tissues of the rabbit and
which is held there most tenaciously. From this point of view it is not
difficult to understand why the new-born animal is provided with so
great an amount of stored-up iron. During the first few da3^s consider-
able hemoglobin is formed, and, if there were not this supply of iron, it
would be necessary for the milk to contain much more iron than usual in
order to satisfy the requirement. A study of the cells of the milk-glands,
however, shows that on the whole they are nearly fitted to furnish a quite
definite secretion. The task of the cells in satisfying the demands of the
young organism is, therefore, lightened by the fact that the offspring
already has this extra supply of iron available. As Hugonnenq ' has
shown in the case of the human foetus, this storing up of iron takes place
chiefly during the last three months before birth. There is nothing
whatever to indicate that the iron is stored up merely because it is a
substance difficult for the young organism to absorb and assimilate.

Compt. rend. 128, 1054 (1899).

386 LECTURE XVII.

The above tables are of interest in another respect. They show that
the iron stores of the suckUng pkis the amount of iron in the milk are,
towards the end of the period of lactation, no longer sufficient for the
formation of the required amount of hemoglobin. The organism is evi-
dently in a state of iron starvation. As soon as fodder rich in iron is
taken into the system together with the milk, the hemoglobin values
increase rapidly. This shows how undesirable it is that the human off-
spring should be restricted to a milk diet much longer than the ordinary
period of lactation (7 to 9 months). The child then requires more iron
in its food.

Iron has, from days of antiquity, always been considered as playing an
especially important part in the nourishment of the human organism,
and chiefly on account of the pathological condition known as chlorosis,
or green sickness. This occurs particularly in young women at, or near,
the age of puberty. The name is derived from the peculiar, character-
istic, greenish-yellow colored skin of such patients. It was early rec-
ognized that this most prominent symptom — together with the paleness
of the rnucous membrane — was to be traced to anaemia, an impover-
ishment of the blood. The disease has alwaj's been combated by pre-
scribing iron. On examining the blood of those suffering from this disease,
it is found that there is a deficiency in the amount of hemoglobin per
unit of volume. It is not, as in forms of antemia caused by loss of blood,
or otherwise, due to the fact that the total number of blood corpuscles is
diminished and coasequently the hemoglobin content, but rather that
the amount of hemoglobin contained in the individual corpuscles is too
small. While in many cases the actual number of red corpuscles remains
normal, still it often sinks considerably. The only interest that this
disease has for us at this place is to the extent that the study of it, and
particularly of the therapeutic action of iron, has cleared up the question
concerning the absorption and assimilation of this metal and of the other
inorganic elements which are required by the organism. The fact that
inorganic iron salts have a favorable action upon chlorosis has been known
for a long time. How the iron acted was not known, — it was merely
taken for granted that it was absorbed and assimilated; i.e., utilized for
the formation of hemoglobin. This view was perfectly plausible as long as
nothing definite was known concerning the manner in which the iron is
held combined in the hemoglobin molecule. If the hemoglobin merely
contains the iron in a loose, salt-like state of combination, it would be
perfectly plausible to think of an assimilation of inorganic iron salts. It
is now known, however, that the iron contained in hematin, a compound
very closely related to hemoglobin, is in a very firm state of combination.
It resists the action of boiling, concentrated potassium hydroxide and
boiling hydrochloric acid. By dissolving it in concentrated sulphuric

INORGANIC FOODS. 387

acid the iron is split off and the hematin is changed into hematoporphyrin.
The assimilation of the iron, therefore, cannot be a very simple process,
and consequently at the time when it was denied that the animal cells
possessed the power of effecting syntheses, it is inconceivable why the
assumption should, nevertheless, have been made that inorganic iron as
such took part in the formation of hemoglobin. This standpoint was
emphasized by G. von Bunge,' who raised the question as to the form in
which the iron is contained in our food supply. Is it present in the
form of simple inorganic iron salts, or as complicated organic compounds?
He prepared first of all from the yolk of eggs, which must of course
contain the iron necessary for the formation of hemoglobin in the blood
of the chick, a compound which by its entire chemical behavior was
shown to contain iron in a very firm state of combination.^ On extract-
ing the yolk of a hen's egg with alcohol and ether, none of the iron
goes into the extract. All of it remains in the residue, which consists
chiefly of albumin a,nd nucleins. From this residue the iron cannot be
extracted by means of alcohol and hydrochloric acid. Since all salt-
like compountls containing iron combined with inorganic or organic acids
can be removed by the above reagents, it is to be assumed that the iron
is held in a closer form of combination than is the case with its ordinary
salts. Bunge next isolated the compound containing iron which was
formed by the action of the juices of the stomach during the digestion
of protein. The part containing iron does not go into solution. It is
indigestible, and corresponds in its entire behavior to that of a nuclein
substance. The iron cannot be extracted by alcohol containing hydro-
chloric acid, but is dissolved out by aqueous hydrochloric acid, the rate
of solution increasing with the concentration of the acid. Although the
iron contained in hematin does not react with the ordinary reagents used
for detecting the presence of iron, namely, ammonium sulphide, and acid
solutions of potassium ferro- and ferricyanides, the iron contained in the
nuclein substance does give these tests. By dissolving the nuclein in
ammonia, and then adding potassium ferrocyanide and acidifying with
hydrochloric acid, a white precipitate is produced which turns blue on
standing. In the case of potassium ferricyanide the precipitate remains
white. On adding ammonium sulphide to the ammoniacal solution of
the nuclein a green coloration is formed, gradually turning black in the
course of twelve hours. Recently this compound, to which Bunge gave
the name hematogen, has been prepared by Hugonnenq and Morel,' and
purified from albumin as much as possible. On analyzing it they obtained

' Verhandl. des 13. Kongresses fiir innere Medizin, p. 133 (1895).
^ Z. physiol. Chem. 9, 49 (1884).
' Compt. rend. 140, 1065 (1905).

388 LECTURE XVII.

the following values: 43.5 per cent C; 6.9 per cent H; 12.6 per cent N;
8.7 per cent P; traces of S; 0.455 per cent Fe; 0..352 per cent Ca; and 0.126
per cent Mg.

It appears that we have here a compound representing a preliminary
stage in the formation of hemoglobin. The method of proving this, how-
ever, is not entirely satisfactory. The conclusion rests largely upon an
elementary analysis. It need hardly be mentioned that this does not
mean much in the investigation of such a highly complicated organic
substance. A quite similar compound has been prepared by G. Walter,'
from the eggs of the carp. Walter found in his preparation 48 . 0 per cent C,
7.2 per cent H, 14.7 per cent N, 0.30 per cent S, and 2.4 per cent P; in
a second product he obtained the values 47 . 8 per cent C, 7.2 per cent H,
12.7 per cent N, 2.9 per cent P, and 0.25 per cent Fe.

There is no doubt that the plants also, to some extent at least, con-
tain iron in the form of complicated organic compounds; and, in fact, it
seems evident that nuclein substances containing iron are present. Plants
rich in iron, especially spinach, are well suited for the preparation of such
products. By a similar treatment to that described for the preparation
of hematogen from egg-yolk, a product relatively rich in iron is obtained,
which, like hematin, is not acted upon by the juices of the stomach,
gives a similar elementary analysis, and shows corresponding chemical
reactions. These compounds are always obtained in an amorphous state,
so that it is difficult to judge of their purity. To attempt to draw any
conclusions as to their identity, or as to the relation of ihese compounds
to one another, would be extremely hazardous.

We do not know the form in which the iron is contained in milk. The
amount present is so small that up to the present time it has not been
possible to isolate any compound containing iron.

Starting with the hypothesis that the iron in our food is present, at
least to some extent, in a state of firm combination with organic material,
Bunge next raised the question as to whether the animal organism is so
constituted that it is able to absorb inorganic iron salts. It was not an
easy question to answer. It was customary to recognize the absorption
of a substance only when it, or one of its related compounds, was found
in the urine. This is not the case when inorganic iron salts are taken
into the system.' Only after subcutaneous introduction is there any
considerable amount of iron to be found in the urine. The fact that when
iron is incorporated into the system in this way it has a poisonous effect
upon the organism, gives further support to the assumption that inorganic
iron salts cannot pass through the intact intestine,' for there has never

' Z. physiol. Chem. 15, 477, 489 (1891).

' R. Kobert: Arch, exper. Path. Pharm. 16, 361 (1883).

' H. Mayer u. Francis Williams: .\rch. exper. Path. Pharm. 13, 70 (1881).

INORGANIC FOODS. 389

been any poisoning observed from taking iron salts, provided the doses and
the manner in which they are introduced are so chosen that large amounts
of iron do not get into the circulation by erosion of the ahmentary
canal. Little by little the view became accepted that iron and the
heavy metals find their principal place of elimination not in the kid-
neys, but in the intestines. It is true that a part of the iron leaves the
body through the urine, for the latter invariably contains small amounts
of iron. The amount, however, is very slight, and is not materially
increased when iron is taken as medicine. Long ago it was suggested
that probably a large part of the iron was eliminated through the intes-
tines, and was, therefore, to be found in the fieces. The fact that after
taking iron into the system, the most of it did actually appear in the faeces,
led to the erroneous conception that no absorption took place. It has
been shown in the first place that subcutaneous and intravenous injection
of iron salts always result in a part of the iron being eliminated through
the intestines.' On the other hand, it may be said that this does not rep-
resent a normal condition. It was indeed conceivable that the organism
seeks to get rid of the iron salts poisonous to it as quickly as possible by
all the means available. The credit of having done the most towards
explaining the absorption and elimination of iron salts belongs chiefly to
Kunkel, Quincke, and Hochhaus.^ These investigators made use of
ammonium sulphide as a reagent for tracing the course of iron in the
tissues;^ in some eases, potassium ferrocyanide was used as well. If, for
example, mice are fed for a long time upon milk which, as we have seen,
contains but Ijttle iron, then, on placing the alimentary canal of these
animals in ammonia and ammonium sulphide, the iron test does not
appear, or at most there is only a slight green coloration.^ The same is
true of the other organs of mice, after they have subsisted upon milk
alone for a considerable period. The best tests for iron are given by the

' Hamburger: Z. physiol. Chem. 2, 191 (1878-79); Lapicque: Arch. Physiol, normale
et pathol. 1895.

' Kunkel: Pfiuger's Arch. 50, 1 (1891); 61, 595 (1895). Filippi: Arch. exp. Path.
Pharm. 34, 462 (1895). Hochhaus u. Quincke: ibid. 37, 159 (1896). Quincke: ibid.
37, 183 (1896). W. F. C. Woltering: Z. physiol. Chem. 21, 190 (1895-96), and Over de
resorptie van ijrerzonten in het spysverteringskanaal, Utrecht, 1895. W. S. Hall:
Arch. Anat. Physiol. 1896, 49; 1894, 455. A. Macallum: J. Physiol. 1894, 186. Abder-
halden: Z. Biol. 39, 113 (1899). A. Hofmann: Virchow's Arch. 151, 484 (1900). G.
Swirski: Pfliiger's Arch. 17,466(1899). Tartakowsky: ifeirf. 100, 586 (1903). Hubert
Sattler: Ueber Eisenresorption u. Ausscheidung im Darmkanal bei Hunden u. Katzen
Inaug. Diss. Kiel, 1904, and Arch, exper. Path. Pharm. 52, 326 (1905). Franz Miiller:
Deut. med. Wochschr. No. 51 (1900) and Deut. Med.-Ztg. No. 30 (1901). Virchow's
Arch. path. Anat. klin. Med. 164, 436 (1901).

= Cf. R. Gottlieb: Z. physiol. Chem. 15, 371 (1891).

* A. Mayer was the first to apply this method (Dorpat, 1850). Later, Perls (Vir-
chow's Arch. 39, 42 (1867)) made use of K,Fe(CN),.

390 LECTURE XVII.

liver and spleen.' The facts are quite different in the case of animals
which have been given iron with the milk. In the stomach there is ob-
tained but little, if any, reaction for iron, while in the duodenum there is
a marked green coloration. Often the reaction is localized to definite
zones. It is frequently found, for example, that merely the top of the
intestinal villi are colored green. If the tissue of the intestine is
examined under the microscope, numerous little kernels containing iron
are to be found embedded in the protoplasm of the intestinal epithelium,
and for the most part these are directly beneath the cuticular borders of
the cells. Now and then tiny leucocytes may be seen laden with innumer-
able little particles of iron. These are noticed in the stroma of the villi.
In the submueosa also, cells containing iron may be noticed once in a
while. In the jejunum, however, it is quite different. Here, as a rule,
the iron reaction is shown only in the solitary follicles and in the Pei/er's
patches. In the ileum, the iron reaction is not, as a rule, very pronounced,
while the caecum and large intestine again give a strong test. Coming
from the intestinal canal, especially the duodenum, lymphatics filled
with cells containing iron may often be seen leading to the mesenteric
glands, which likewise show a pronounced iron reaction. The liver and
spleen now give a very strong test, and evidently these organs serve as
storage places for iron salts. The muscles and bone-marrow, etc., like-
wise show a green coloration when tested with ammonium sulphide.

Before going farther, we wish to emphasize the fact that the observed
conditions which were obtained with mice, rats, rabbits, guinea pigs, dogs,
and cats, are not to be attrilauted to an irritating effect of the iron. It is
perfectly clear that if the epithelium of the intestines is mjured in any
way, the absorption relations which normally take place in the intestines
will be considerably affected. Such irritation has indeed often been
observed in experiments where large doses of iron salts were fed to animals,
but in the experiments now in question the amounts of iron were very
small. Thus, rats were given but 0.4 to 0.5 milligram Iron in the course
of a day, rabbits 4 milligrams, guinea pigs 2 to 3 milligrams, dogs .3.5 to
4 milligrams, and cats 4 milligrams per day in the form of ferric chloride
added to the milk. This small amount of iron was taken up gradually
in the course of the day.

Now what is the significance of the above discoveries? The simplest
assumption is that iron begins to be absorbed in the duodenum, and is then,
to some extent at least, carried first of all to the lymphatic ducts. Obser-
vations made by Gaule ^ make it seem probable that part of the iron is
carried away to the thoracic duct, and thus reaches the circulation. It is

' Abderhalden : loc. HI.

' Deut. med. Wochschr. 1896, Nos. 19 and 24.

INORGANIC FOODS. 391

certain that a part of the absorbed iron is also conducted to the portal
vein of the liver. The latter organ forms one of the chief storage places
of iron salts. The spleen also absorbs considerable iron. It is probable
that the lymph carries some of the iron back to the intestines, and is
eliminated through the caecum and large intestine. Evidently the leuco-
cytes play an important part in the processes of taking up and trans-
porting the little particles of iron, for they are often observed laden with
iron particles, both in the paths of absorption and in those of elimination.
There is no doubt that a large portion of the iron introduced into the
organism is again eliminated through the intestines. It is at present
uncertain as to what parts of the intestines share in this elimination.
This is particularly true of the small intestine, which perhaps partici-
pates in both the absorption and elimination of iron. We must not
forget, above all, that microchemical reactions have a limited value.
They serve merely to give us a qualitative picture of the activity of
definite compounds, but never give us much idea as to the amounts of
substances entering reactions. Moreover, the method used in the above
experiments for detecting the presence of iron qualitatively was not
entirely satisfactory. We have already mentioned, for example, that
the iron in hematin does not react with the ordinary reagents used in
testing for the presence of iron, and that hematogen only gives a notice-
able reaction after the solution has stood for some time. It is easy to
conceive that the animal organism may contain its iron firmly bound in
other compounds besides hematin, so that in any case the mere fact that
we do not detect the presence of iron by a qualitative test does not prove
that the tissue tested does not contain this element. On the other hand,
the fact that we do not get these iron tests in cases where other methods
(examination of the ash) show that it is actually present, does not give
any reliable information concerning the way that the iron is held in com-
bination. We have already seen that colloids play an important part in
the animal organism, and that they are apparently capable of preventing
certain reactions from taking place. Thus we found that the formation
of precipitates was often prevented, even in caises where the reaction had
actually taken place. The precipitate is not seen, merely because the
internal friction between the colloid particles prevents the tiny parts of
insoluble substance formed from uniting to cause a visible precipitate.
This point is all too frequently lost sight of in scientific work. In many
cases the statement that iron is present in a firm state of " organic "
combination is not justifiable.

It is, we regret to say, at present impossible to follow with sufficient
accuracy the question of the absorption of iron and the distribution of
the absorbed iron among the separate organs. The amounts of iron
absorbed are so small that with our present methods it is entirely out of

392 LECTURE XVII.

the question for us to attempt to picture the course of the iron through
the organism, and to decide how much inorganic iron is absorbed, and
for how long.

The conditions which we meet with in the case of iron as regards its
absorption and eUmination are similar with other elements. Apparently
other inorganic elements are taken up in about the same way and are
similarly eliminated. Thus we know that if we add lime ' to the food, a
part of it is eliminated in the urine, whereas a considerable portion is
deposited in the large intestine. Inorganic elements which are not usually
found in the organism follow the same course. Thus, Steinfeld ^ found
that subcutaneous injection of bismuth into birds caused the vermiform
process and large intestine to be colored black. The relations of elimi-
nation are similar with mammals. Thus Steinfeld found on repeating
this experiment with dogs and cats that the lymph vessels of the intestines
were filled with a black substance.

The fact that when iron was administered, a part of the dose was
absorbed in the duodenum, and then again eliminated at the end of the
intestine, does not tell us what happens to the iron that is contained in
our ordinary food. This had to be determined by a direct experiment.
It was found ' that animals fed exclusively upon meat or exclusively upon
vegetables gave exactly the same reactions for iron in the intestines and
in the tissues as those which were dosed with iron salts. The behavior of
the hemoglobin and hematin was particularly interesting. Both of these
compounds contain iron in a form which renders it impossible to detect
it by the ordinary chemical reactions. If now a rat was fed with milk
containing either hematin or hemoglobin, while another rat was fed with
milk alone, then the alimentary canal of the latter would not give any
iron reaction, whereas if the intestinal tract of the first rat was placed
in a solution of ammonia and ammonium sulphide, an intense green color-
ation appeared at once. Thus the iron has been loosened from its state
of combination in hematin; it has evidently become ionized. This shows
it is extremely probable that unchanged hematin is not absorbed as such.
The formation of hemoglobin in the animal organism is in all cases the
result of a synthesis or a change in the way in which the iron is combined.
This assumption cannot at present be verified definitely, because, as we
have mentioned, it is possible that besides the compounds in which we are
able to detect iron by the ordinary reagents, there may be others present

' R. W. Raudnitz: Arch, exper. Path. Pharra. 31, 343 (1893); J. G. Rey: Deut. raed.
Wochschr. No. 35, 569 (1895). G. Riidel: Arch, exper. Path. Pharm. 33, 79 (1894).
J. G. Rey: ibid. 35, 295 (1895).

' Dissertation, Dorpat, 1884. Meyer and Steinfeld; Arch, exper. Path. Pharm. 20,
40 (1886).

' Emil Abderhalden: loc. cit.

INORGANIC FOODS. 393

which are capable of absorption in which the iron is too firmly bound to
react with such chemicals and thus escapes microchemical detection. It
is at least decidedly interesting to know that even under normal conditions
of nourishment the tissues of the animal organism always contain iron in
compounds which permit its detection by means of the ordinary reagents.

The mere fact that inorganic iron salts artificially added to the nourish-
ment are capable of absorption and deposition in the tissues does not
show by any means that this iron is actually assimilated. It would be
insufficient to consider the assimilation of iron from the standpoint of blood
formation alone. There is no doubt that iron forms an integral part of
all cells and tissues of the body. Certainly, this iron in the tissues is just
as essential for the normal functions of the separate organs as is the case
with the iron in hemoglobin. Some idea of these relations may be ob-
tained from the values cited for the iron in the tissues of rabbits during
the period of suckling. It is evident from these values that the tis.sues
hold with great tenacity to a definite minimum amount of iron, even
when the hemoglobin content of the entire organism is so far diminished
that strong anaemia results. At present we have no definite information
as to the part played by this iron in the tissues. We do not even know
what amounts are present in the separate organs. It is likewise difficult
to decide what portion of the total iron has been introduced and what
portion is about to be eliminated. Thus it is easy to realize how the
question concerning the assimilation of iron has been covered by that
of the formation of the hemoglobin.

At the present time it has never been definitely decided whether the
iron introduced into the system in the form of inorganic salts takes part
in the formation of hemoglobin or of hematin. We find ourselves here,
as is the case with many other biological experiments, confronted with
the condition that one and the same discovery may be used, to support
entirely opposing views. The old experience of physicians, that iron pills
combat chlorosis, can be explained by the assumption that the iron in the
pills goes to form the hemoglobin. On the other hand, judging from
analogy with other observations, it is perfectly plausible to assume that
the iron in the pills merely has an indirect effect in that it excites those
organs which take part in the formation of blood into increased activity.
First of all, it is necessary to determine whether chlorosis is actually
caused by a deficiency of iron.

Let us see how much iron there is in the human organism. A mouse
contains 100 milligrams iron per kilogram of its weight, a guinea pig con-
tains but 52 milligrams per kilogram, and a rabbit about 46 milligrams.
If we assume that the last value is about right for a human being, then we
would have in a person weighing 70 kilograms about 3.2 grams of iron.
The question is How much iron does the human organism eliminate? Star-

394 LECTURE XVII.

vation experiments on man show that there is 7 to 8 milligrams of iron
eliminated daily from the intestines.' Now a liter of milk contains about
2.3 milligrams of iron. With milk alone as food, therefore, it would be
necessary to drink at least four liters per day, for during starvation the
organism is extremely economical with its stores, and the amount of
iron then eliminated represents a minimum. Certain of our foods, such
as white bread, rice, cherries, apples, etc., are very deficient in iron.^ It
is, therefore, easy to believe that people subsisting exclusively upon such
nourishment will have impoverished blood; and, as.a matter of fact, many
cases of chlorosis may be accounted for in this way. The practising
physician, however, knows of many cases of chlorosis in which unquestion-
ably enough iron has been taken into the system to satisfy the ordinary
requirements. In such cases we are forced to assume that the function
of absorbing iron has in some way become disturbed, or else that the iron
compounds taken into the system for some reason are not utilized by the
organs which serve to form the blood. As a matter of fact, disturbances
in the function of the intestinal tract have been observed frequently in
chlorosis. Even in such eases, however, which are, by the way, excep-
tional, it is not known whether these intestinal disturbances are of a
primary nature, or whether they are not rather a result of the impov-
erished blood and the disturbances of nutritional relations which result
therefrom.

The attempt has been made to decide by means of experiments upon
animals what the relations are between iron introduced into the organism
in an inorganic form and the formation of the blood. Two different
methods have been tried. We have seen that at the end of the lactation
period the suckling possesses a low hemoglobin content, and that this
increases rapidly as the animal begins to partake of food richer in iron.
If, on the other hand, the animal is kept for a prolonged period upon a
milk diet, a marked ancemia results. That this is, as a matter of fact, the
case,' is shown by comparing the amounts of hemoglobin in animals
which have from birth been fed only upon milk, with that of animals
which, at the end of the suckling period, have passed over to a diet richer
in iron (vegetables). Now the addition of inorganic iron salts to the
milk has no effect upon the absolute amount of hemoglobin contained in
the animals, nor upon the amount per kilogram of the animal's weight.
The added iron, however, is found to have a remarkable effect in acceler-
ating the growth of the animal.^

' Lehmann, MiiUer, Munk, Senator, and Zuntz: Virchow's Arch. 131, Suppl. 1, pp. 18
and 67 (189.3).
2 Cf. p. 380.

' Emil Abderhalden: Z. Biol. 39, 193 (1899).
« Emil Abderhalden: Z. Biol. 39, 483 (1899).

INORGANIC FOODS. 395

A second method ' for deciding the question concerning the afssimila-
tion of iron is to withdraw equal quantities of blood from two animals of
the same age and, as far as possible, of the same condition of nourishment,
and then to compare the regeneration of blood in one case where the
animal is fed entirely upon milk, and in another where iron is added to the
milk. Here all authors agree that the animal to which the inorganic
iron has been fed is able to replace the lost blood much more quickly
than the other animal to which only the iron contained in milk is avail-
able. The disagreement in this result with that obtained in above experi-
ments may perhaps be due to the different conditions of the animals in
the two types of experiments. In the first instance, animals were chosen
whose iron stores at the beginning of the experiment were in a quite
exhausted state. All of the iron in the different, tissues was brought
down to the lowest limit. The animals in the second cases were in an
entirely different condition. These possessed a considerable supply of
stored-up iron, and would have been able to supply the loss in hemoglo-
bin iron from these stores alone. That the addition of inorganic iron
exerts a favorable action might be in fact explained on the assumption
that it excites the action of the blood-forming organs. We must admit,
however, that such an explanation does not seem well founded in this
case, for it would seem probable the large loss of blood would of itself
serve to incite the organs which take part in the formation of blood into
increased activity. On the other hand, it is conceivable that the inorganic
iron replaces the iron in the tissues, so that the latter is left free to take
part in the formation of hemoglobin. No one has ever been able to prove
this directly, but it seems to be a likely explanation, as we shall see from
what follows.

Franz Miiller - hoped to settle this question by an examination of
bone-marrow, one of the principal places where the red blood-corpuscles
are formed. Miiller fed dogs, taken immediately upon the completion of
the lactation period, on the one hand e.^clusively with milk, and on the
other with milk and iron. After subsequently killing the animals, he
found that the bone-marrow of the animals fed with iron contained con-
siderably more nucleated, red blood-corpuscles than the animals fed
with milk alone. Again, this discovery may be interpreted in two differ-
ent ways. We may assume that the inorganic iron takes part directly
in the formation of hemoglobin, or, on the contrary, that it merely exerts
an indirect action.

When we take into consideration all of the different experiments that
have been performed in this connection, we must unhesitatingly admit
that it has never been satisfactorily proved whether the iron taken into

' Kunkel: Pfluger's Arch. 61, 596 (1895). Eger: Z. klin. Med. 22, 335 (1897).
' Loc. cil. Cf. also A. Hofmann: loc. cit.

396 LECTURE XVII.

the system in an inorganic form can take part in the formation of
hemoglobin. The question then naturally arises, Have the proper means
been found for solving this problem ? Up to this point, in discussing the
formation of hemoglobin, we have considered but one component of hema-
tin; namely, the iron. It seems hardly justifiable to limit the entire dis-
cussion to this one component. We know that the chemical composition
of hematin is very complicated.' M. Nencki and J. Zaleski ^ have sug-
gested the following structural formula for hemin, the hydrochloric acid
ester of hematin:

CH2 CH2

CH_^/\ CH— (0H)C/\^1 CH

phII II lip pll II IICH

NH CH2 1 ICH2 NH

FeCl 0

CH2 CH2

CH_2^'^C C/\— CH

ChII II^JJCH— (OH)cll^^J,^ JCH

NH CH2 CH2 HN

We introduce this formula here merely to show how complicated its
synthesis must be if the animal cells are to build it from inorganic iron.
The formula also shows that obviously the iron itself does not play such
an all-important part in the synthesis; that is to say, it is probably not so
very difficult to introduce the iron into the molecule. It is infinitely
more important to know whether the organism has other organic material
available for the formation of the hematin.

In the above formula we see that two hematoporphyrin molecules
(the iron-free cleavage-products of hematin) are held together by an iron
atom. The formation of hematin, therefore, depends just as much upon
the presence of material from which hematoporphyrin can be made as upon
the presence of iron. Now we know, as will be explained subsequently
in detail, that hematophorphyrin is closely related to chlorophyll,' for
similar decomposition products are formed from each of these two com-
pounds. This suggests the thought that chlorophyll can perhaps be
brought into relation with hematin, and thus with the formation of hemo-
globin. There is absolutely no doubt that these compounds are closely
related to one another. The fact that both, as we shall see later on,
possess similar biological functions is sufficient to explain this relation-
ship, although, as a matter of fact, the task of chlorophyll is quite different,
as far as our knowledge goes, from that of hemoglobin. Now, inasmuch

' See Lecture XXIV.
2 Ber. 34, 997 (1901).
' L. Marchlewski : Die Chemie des Chlorophylls (1895).

INORGANIC FOODS. 397

as the original source of all substances in the animal organism can be
traced back eventually to the vegetable kingdom, the thought suggests
itself that chlorophyll serves as the building material in the formation of
hemoglobin. The herbivora devour this compound in considerable
quantities, and, on the other hand, the carnivora receive considerable
amounts of hemoglobin with their nourishment. Unfortunately, we
know nothing definite concerning the transformations which take place
with these two products in the alimentary canal. Of hemoglobin we now
know that in its absorption it is so changed that the iron in it becomes
detectable with ordinary chemical reagents. According to our experi-
ences as regards the significance of digestion, it is perfectly possible that
chlorophyll and hemoglobin are broken down into similar products, and
thus that the organs which form the blood have practically the same
kind of building material presented to them in each case. The actual
amount of chlorophyll and hematin available does not need to be very
large. Unfortunately, we do not know how much hemoglobin is formed
daily, or how much is decomposed, so that we have no data to judge as to
the normal extent of hemoglobin formation.

We arrive at this line of thought because evidently the synthesis of
hematin is a very complicated one, and we are aware of no material from
which it could be formed apparently so readily as from chlorophyll or its
decomposition products.' At all events, these views do not by any means
imply that inorganic iron cannot take part in the formation of hemo-
globin. Even if we grant that the nucleoalbumins, or nucleoproteids,
serve as the building material of hemoglobin, it does not seem to us that
this makes the direct assimilation of iron impossible, for these substances
contain iron in a relatively loose state of combination. The formation
of hematin from albumin compounds containing iron must necessitate
quite considerable molecular rearrangements in order to get the iron in
the state of union known to exist in hematin. Now the question arises
as to whether milk possesses the material, other than iron, in sufficient
quantity for the formation of hematin. We have a perfect right to doubt
this. It would be perfectly useless to provide the organism with a suffi-
cient amount of hematoporphyrin, the principal component of hematin,
when an adequate supply of iron, the minor constituent, is not available.
It is far more probable that milk contains these two constituents in about
the same relative amounts as in hematin. It is consequently perfectly

' Perhaps the cleavage products of protein come into consideration as building
stones. Both proline and glutamic acid could furnish material for the pyrrole ring.
Plant albumins contain large amounts of glutamic acid. The reserve albumins are rich
in the above-mentioned amino acids. Perhaps this has something to do with the for-
mation of chlorophyll, and it is perfectly possible that the animal organism chooses the
same way for preparing its hematin.

398 LECTURE XVII.

possible that in the above-mentioned experiments in which iron was added
to the milk, there was not much effect on the amount of hemogloljin
formed, because there was an insufficient supply of the other building
material out of which hemoglobin is formed. Again, we have up to this
point left out of consideration the fact that besides hematin, there is
another component of hemoglobin, namely globin, which is an albumin
substance of highly complicated structure. The formation of the hemo-
globin molecule is complete only after the hematin has united with the
globin molecule.

Enough has been said to show that the formation of hemoglobin does
not solve the question as to the part that iron plays in its formation. The
kernel of the whole question has not yet been attacked. We cannot hope
for a solution of the problem until we understand clearly the formation of
hematin. The mere fact that the addition of iron to nutriment poor in
iron does not have any distinct influence upon the formation of hemo-
globin, in no way speaks against the participation of inorganic iron in the
synthesis of hemoglobin in the case of normal nutrition, but it indicates
that the other building material is wanting as well as the iron. Further-
more, the fact that when the animal passes over to a form of nourishment
richer in iron, there is a rapid increase in the extent of the hemoglobin
formation, is explained not only by the increased amount of iron in the
food, but as well by the fact that the other material required for the pro-
duction of this substance is likewise available to a much greater extent.

Let us now return to chlorosis. We must first of all emphasize the fact
that the anemia produced by an exclusively milk diet, or bj^ loss of blood,
has nothing whatever to do with the disease in which there is an impov-
erishment of the blood. In the case of typical chlorosis, the composition of
the blood is abnormal in spite of the fact that there has been available a
sufficient supply of substances which take part in the formation of blood.
It is characteristic of the disease that it occurs in the full-bloodedness of
the female organism's development, in the years of puberty, and it
gradually disappears without any change in the nourishment sufficient
to account for the correction of the disorder. One gets the impres-
sion that demands are suddenly made upon the blood-forming organs
which it is not able to satisfy. It would be easy to conceive that the
blood losses brought about by menstruation are the cause of the increased
demands upon the hematopoietic system. It has been shown, however,
that the amount of iron lost in the flow of blood during menstruation ' is
very slight, and need hardly be taken into consideration. Bunge, from his
observations that the suckling was born with a store of iron, made the
suggestion that the organism, in order to be able to give up this supply of
iron, must begin to store up iron before the time of conception, so that it

' Hoppe-Seyler, Brodersen and Rudolph: Z. pliysiol. Cheni. 42, 545 (1904).

INORCxANIC FOODS. 399

would have, aside from its food, a sufficient supply of iron during preg-
nancy. In order to test this hypothesis Bunge ' made a number of experi-
ments, and determined the amount of iron in the chief storage place, the
liver, in both male and female cats and dogs, but the results obtained were
not altogether harmonious.

Although we are not in a position to assign a cause for chlorosis, still it
is perhaps possible to explain the curative effect of the iron preparations.
If we, however, study closely the whole process of the hemoglobin forma-
tion, it seems to us less probable that the iron preparations added to the
nourishment actually take part directly in the making of blood. It would
be easy to understand such an action, if chlorosis were actually caused by
a deficient supply of iron. We are perfectly certain, however, that in the
majority of cases this is not true. We have every reason to presume that
our nourishment in general, besides containing sufficient iron, likewise
contains enough of the other substances required for the formation of
hemoglobin. Just as the tissues of the bones in richitis are not capable
of assimilating lime, so, evidently, the tissues of the hematopoietic organs
are not able to utilize the material which forms the building stones of
hemoglobin. It has been suggested that the iron added in the form of
inorganic salts exerts an irritating effect upon these orgaas, and urges them
into renewed activity. The unsatisfactory character of such an explana-
tion is evident. It assumes that the iron fed to the body in an inorganic
condition, behaves otherwise than that contained in "organic " com-
pounds. We have, however, at present no insight into the waj's and
means by which absorbed iron reaches the circulation, nor as regards the
form in which it is present in the different fluids and tissues. We only
know that the iron in the organs can be detected by means of the ordinary
chemical reagents, irrespective of whether the animal has been fed with
milk and inorganic iron, or milk and, say, hemoglobin. Even while these
observations do not justify the assumption that the different iron com-
pounds all behave alike after reaching the intestine, — i.e., that they may be
changed into the same state of combination, — still, on the other hand, we
have no justification for the assumption that the animal organism distin-
guishes between iron that is fed in an inorganic condition from "organic"
iron. According to all our general conceptions of the process of digestion,
it appears to us as extremely probable that even the formation of hematin
is preceded by a deep-seated decomposition. In this case the animal
organism unquestionably breaks down and again builds up. If we look
at the formula of hemin given above, it seems to us as highly improbable
that in general " organic " iron is necessary for the synthesis of
hematin. Yes, in fact, we can even imagine that the disease of chlorosis
actually depends upon the fact that the cells of the body are no longer

' Z. physiol. Chem. 17, 78 (1893).

400 LECTURE XVII.

capable of making use of iron which is supplied in the form of highly com-
plicated organic compounds, or at least are unable to convert them into
such a state that they can be utilized for the formation of hematin. Per-
haps the preparatory decomposition in the alimentary canal has not taken
place, and thus the iron may circulate to some extent in the tissues, but in
a form from which the blood-forming organs are not able to set it free.
From this point of view, it is easy to understand the favorable action of
inorganic iron salts. Here we intentionally break away from the older
idea that the animal organism is dependent upon the nature of the food
that it receives. It is far more important that it receives all the materials
that it requires. The way these elements are originally combined in the
food is more or less a matter of indifference, provided they are susceptible
of decomposition. It is indeed this far-reaching decomposition and the
renewed construction which makes the animal organism to a considerable
extent independent of the kind of nourishment it receives. To be sure,
we must admit that apparently the animal cells are not capable of utilizing
certain kinds of combinations. Thus, it is improbable, for example, that
it is capable of constructing cholesterol, and perhaps not hematoporphyrin
from its simplest components, although it is precisely here that we meet
with the possibiUty that certain decomposition products of the albumins
may be utilized for the syntheses. At all events, we are not justified in
believing that hematogen and similar substances are necessarily direct
preliminary stages in the formation of hemoglobin. It is indeed possible
that these compounds contain all the necessary material for the formation
of the red pigment of the blood, although this has never been proved
directly.

The theory we have thus developed concerning the action of inorganic
iron salts is not necessarily correct. By the addition of iron preparations
to the food, we increase the iron supply for the entire organism. It is
conceivable that this results in an indirect action upon the organs which
produce the blood. We know that widely different organs are intimately
related to one another in their metabolism and in the exercise of their
functions. In this connection, we need refer only to the regulation of the
sugar-content of the blood by means of the liver. Similarly the amount
of hemoglobin in the blood is a very constant quantity. Evidently we
have here another case of regulation. On the other hand, we have seen
also that the different ions exert a quite specific effect, and that it is,
indeed, possible for the predominance of one ion to cause a certain specific
action. It is conceivable that the heaping up of iron in the cells of the
body, and perhaps specially in the cells of certain organs, may give a new
impulse to the organs which participate in the formation of blood.

The action of the " inorganic " iron was formerly attributed to a pro-
tective effect. Thus the sulphur in alkaline sulphides, instead of com-

INORGANIC FOODS. 401

bining with the more complicated organic compounds containing iron,
was beheved to unite preferably with the iron of inorganic compounds.
The iron in the nutriment would thereby be protected, and remain in the
system instead of being eliminated as sulphide of iron. Since it has been
shown, however, that even inorganic iron salts are absorbed, and it has been
proved, moreover, that alkaline sulphides are not present in the stomach
and small intestine, this protective theory may well be discarded.'

From the experiments that have been cited, it is perfectly clear that in
reality we Icnow very little concerning the cause of chlorosis, and especially
concerning the action of iron preparations. On account of the complexity
of the processes involved, we can hardly expect at present to understand
perfectly the relations between the iron and the pathologj^ of the blood
formation. Above all, we need to know more about the proce.ss of blood
formation, and especially as regards the formation of hemoglobin itself.
At the same time the solution of the problem of the formation of hemo-
globin does not by any means necessarily solve the whole problem of the
formation of the blood. We then have to consider the formation of the
blood corpuscles. The stroma of the blood corpu.scles must also be built
up, and, in such a way that it can take up the hemoglobin. A long chain
of processes leads from the separate building materials of hemoglobin,
the iron and the organic compounds, to the finished blood corpuscles
capable of exerting their important functions. The chain may be broken
at many places, and thereby the whole process of blood formation dis-
turbed. This so infinitely complicated problem has been attacked only
from one side, that of the iron. Undoubtedly iron is indispensable in the
formation of blood, but equally indispensable are all the remaining and
building materials which are far more complicated, — ^the hematin, hemo-
globin, and even the corpuscles themselves.

This is not the place to discuss whether iron preparatioas actually
do have any effect upon chlorosis. It is indeed conceivable that it is the
dietetic and hygienic measures that are taken that are alone effective in
iron therapeutics. We have followed chlorosis and iron therapeutics
thus far only in the hope that we would be able thereby to get some idea
of the relation of iron to the formation of blood. On the contrary, the
above conclusions apparently lead us to the opinion that chlorosis
itself is not difficult to understand, — we can account for its appearance
if we assume that the function of the organs producing blood are in any
way disturbed, — but on the other hand, the fact that inorganic
iron preparations ' are successful in combating the disease rather stands

' Cf. Kletzinsk-y: Z. Ges. Aerzte Wien X, II, 281 (1854). Hannon: Presse medical
(1861). Woltering: loc. cit. G. von Bunge; Lehrbuch d. phys. chemie, p. 94 (1894).

' Nearly all of the " organic " iron preparations on the market belong to this class
of iron salts, for they contain the iron in a loose state of combination.

402 LECTURE XVII.

in the way of the general conception which prevails concerning its cause.
According to the theory, however, that it is not the function of synthesizing
the material which is disturbed, but rather the function of decomposing
the material containing the iron, it is easy to understand the favorable
action, of iron salts, for in them the organism receives material which for
some reason it cannot itself obtain. At all events, in cases of iron thera-
peutics it should be borne in mind that hemoglobin cannot be formed from
iron alone, so that care must be taken to supply the remaining material
necessary, in the form of meat, eggs, and green vegetables.

The importance of iron for the tissues has been in the past almost
forgotten in the discussion of the relations of iron to the formation of
blood. Here again iron plays an important part, Wt unfortunately we
now know but little in regard to the way it is combined in the tissues and
cells. It evidently occurs in different forms. Thus, iron compounds
have been prepared from the liver, spleen, and muscles of man by P.
Marfori ' and 0. Schmiedeberg.^ According to these investigators, the
iron is present in much the same state of combination as in hematin. The
substances thus prepared were apparently absorbed by dogs after they had
been starved, or fed upon a diet free from iron. The exact nature of
these iron compounds has by no means been fully explained.

Copper plays, in the case of invertebrates, a similar part to that
taken by iron in the vertebrates.^ It is found combined with albumin.
The compound which corresponds to hemaglobin is called hemocyanin.
It is found especially in many mollusks and crustaceans. The blood of
the cephalopoda has been examined chiefly in this connection. The
arterial blood of these animals is blue, the venous blood colorless. In
certain other mollusks {Pinna squamosa, Doris, Patella, Chiton) manga-
nese '' replaces the copper.

We have, up to this point, failed to mention certain inorganic salts
which occur in milk, and unquestionably are indispensable foods. Thus,
milk always contains some magnesium. This element forms an integral
part of plant and animal cells and also of the animal fluids, blood and
lymph. The amount of magnesium in milk is in general relatively small.
Its function in the animal economy is not yet definitely known. ^ Appar-

' Arch, exper. Path. Pharm. 29, 212 (1891), and Arch. ital. biol. 21, 1 (1894).

= Arch, exper. Path. Pharm. 33, 102 (1894); cf. Filippi: ibid. 34, 462 (1895).

^ a. Harless: Miiller's Arch. 1846, 148. Schlossberger: Ann. 102, 86 (1857).
FrM^ricq: Arch. Zool. exp^r. 7, 535 (1878); Compt. rend. 87, 996 (1878). Dh^r^:
Compt. rend. soc. biol. 52, 458 (1900). Griffiths: Proc. Roy. Soc. Edinburgh, 18, 288
(1890-91); 19, 127 (1892); Compt. rend. 114, 496 (1892). Cf. Otto v. Furth: Vergleich-
ende chemische Physiologie der niederen Tiere, p. 74, Jena, 1903.

* Griffiths: Compt. rend. 114, 840 (1892); 115, 259 and 474 (1892); 116, 1206 (1893),
and Respiratory Proteids, London, 1897.

' Cf. Lecture XVI, p. 354 et. seq.

INORGANIC FOODS. 403

ently it bears about the same relation to calcium as potassium to sodium.
J. Malcolm ' has shown that the introduction of soluble magnesium salts
into adult animals causes a loss of calcium. In growing animals it tends
to prevent the taking up of calcium. Soluble calcium salts apparently
have no effect upon the elimination of magnesium salts. In osteomalacia
we have also come to recognize a certain antagonism between these two
kinds of salts.^

Fluorine^ likewise occurs in small amounts in milk, and forms a regular
constituent of bones and teeth, besides being found in the blood.* In
spite of the small amount present it cannot be disregarded. For this
element, as well as for all others, the Law of the Minimum holds.

Phosphorus is of much greater importance both for the growing and
adult organism. We find phosphorus in the cells in the form of very
important compounds, namely lecithin, the nucleins and nucleoalbumins.
We know, furthermore, that phosphorus combined with the alkaline
earths forms one of the most important constituents of the human skele-
ton, and is also present in the same form in other tissues. Phosphorus is
present in milk, partly in organic combination, as in casein which belongs
to the group of nucleoalbumins, and partly as inorganic salt. Milk also
contains some lecithin. At present it is not known exactly how the
phosphorus is distributed between these different compounds in the
different kinds of milk. Apparently the amount of lecithin- present is
not very large.

There is no reasonable doubt that the living organism can utilize
phosphoric acid directly in the formation of lecithin. It is similarly
possible that it forms a part of its nucleins from the latter substance.
The fact that the animal organism can form lecithin from phosphates
without difficulty is apparent from the experiments already cited of
Miescher upon salmon.^

Phosphorus is especially important in the construction of nervous
tissue. The brain of a new-born infant weighs about 400 grams. This
weight is doubled during the period of lactation. According to Schloss-
mann's computations," the nursling assimilates during this period for
the building up of its central nervous system alone about 0.75 gram of
phosphorus. The skeleton requires much more of this element. In
fact, if we estimate the total amount of phosphorus required by the infant
during the first year of its life, we shall find that it amounts to from 50 to

' J. Physiol. 32, 18.3 (190.5).

' Cf. Lecture XVI, p. .377.

"> G. Tamraann: Z. physiol. Chem. 12, 325 (1888). S. Gabriel: ibid. 18, 281 (1894).

* J. Nickles: Compt. rend. 43, 885 (1886); Tammann: loc. cit.

'■ Cf. Lecture XVI, p. 351.

' Med. Klinik. No. 11 (1905); Arch. Kinderheilk. 40, 1.

404

LECTURE XVII.

60 grams. The amount of phosphorus required in the food is naturally
even far greater, because in the above estimate it was not taken into con-
sideration that phosphorus is constantly being eliminated in the form of
phosphates. In one liter of human milk there is present 0.19 gram
phosphorus, ass's milk contains 0.76 gram, cow's milk 0.79 gram, and
goat's milk 0.96 gram. Human milk, therefore, is deficient in phos-
phorus; it contains less than any of the other kinds of milk which have
been analyzed. This is a remarkable fact, for we know that the human
offspring is able to construct, while still nursing, a nervous system which
is but slightly developed. Compared to human milk, that of the above
animals is extremely high in phosphorus. There must be some reason
for this difference. Bunge, who noticed this fact in his analyses of differ-
ent kinds of milk, compared the percentage composition of the ash with
the rate of development of the species.' It is to be assumed a priori that
an animal which develops rapidly will require more building material
than one whose development is slower. If we compare the time required
by the suckling to double its weight at birth with the amounts of albumin
and ash — perhaps the most essential constituents for the formation of
the tissues — • contained in 100 parts of milk, it is evident at a glance that
the amount of these increases in proportion as the development of the
animal is rapid. This is shown by the following table :^

Days Re-
quired to
Double

Weigiit.

100 Parts by Weight of Milli Contain:

Species.

Albumin.

Ash.

Lime.

Phosphoric

Acid.

Man

Horse

Cow

Goat*

Sheep*

Pig'

Cat'

Dog',*

Rabbit '

180
60
47
22
15
14

9i

9

6

1.6
2.0
3.5
3.7
4.9
5.2
7.0
7.4
14.4

0.2

0.4

0.7

0.78

0.84

0.80

1.02

1.33

2 50

0.03
0.12
0.16
0.20
0.25
0.25

0.45
0.89

0.05
0.13
0.20
0.28
0.29
0,31

0,51
0.99

The composition of the milk of a single species is by no means constant.
The amount of albumin and ash diminishes with the age of the suckling.
This likewise has an effect upon the rate of growth as shown by the fol-
lowing values:^

' Fr. Proscher: Z. physiol. Chem. 24, 285 (1897).

» Abderhalden: ibid. 27, 594 (1899).

= Abderhalden: Z. physiol. Chem. 26, 487 (1899).

* Ibid. 27, 408 (1899).

' Ibid. 27, 457 (1899).

INORGANIC FOODS.

405

One hundred parts by weight contain: (a) Before the animal has
doubled its weight at birth:

Species.

Total
Protein.

Pig ■

Sheep

Goat

3.71
4. OS
2.91

1.65
0.80
0.76

5.36

4.88
3,67

6.32
9.29
4.33

3.19
5.04
3 61

0.105
0 097
0 130

Species.

Na^O.

CI.

FejOj.

CaO.

MgO.

P205-

Total Ash.

Pig . ■ .
Sheep . .
Goat . .

0.082
0.086
0.062

0.083
0.129
0.102

0.004
0.004
0.004

0.268
0.245
0 197

0.017
0.015
0 015

0.329
0.293
0 284

0.871
0.841
0 771

(b) After the animal has doubled its weight:

Species.

Casein. Albumin.

Total
Protein.

Fat. Sugar. Kp

Pig •
Sheep
Goat

3.23
4.07
2.56

1.06
0.52
0 58

4.29
4.59
3.14

7.21
9.44
2.93

3.71
5.22
3.92

0.099
0.096
0 133

Species.

NajO. CI.

Fe^Oj.

CaO.

MgO.

PjOj.

Total Ash.

Pig ■ • •
Sheep . .
Goat . .

0.074
0.085
0 062

0.067
0.121
0.111

0.004
0 004
0.004

0.241
0.235
0.199

0.014
0.015
0.016

0.300
0.281
0.285

0 783
0 809
0.784

During lactation the albumin content of milk diminishes gradually,
while substances such as sugar and fat, which are less essential as build-
ing material, but are rather to be regarded as sources of energy and heat,
tend to increase in amount.

The remarkably small amount of phosphoric acid contained in human
,milk is nevertheless sufficient for the development of the child, although
this takes place much more slowly than is the case with most mammals.
The above relations between the rate of growth and the composition of
the milk make it perfectly apparent how difficult it must be to replace one
kind of milk with that of another species. Evidently if the new milk
contains any constituent in amount less than the required minimum.

406 LECTURE XVII.

there will be, necessarily, disadvantageous results. The value of any milk
substitute should in no case be determined by the fact that it contains
all of the elements required, nor by the fact that it contains in abundance
something (e.g., albumin, lime or phosphorus) which we are accustomed
to regard as especially essential. It is of chief importance that there is
nothing present in quantity below the required minimum. Even though
a milk substitute may be rich in phosphorus, it may be of but little value;
for, in order that the cells may utilize this phosphorus, it is necessary that
a sufficient amount of certain other substances should be present. This
shows where the greatest emphasis is to be placed. There is no question that
the unsuccessful results obtained in the artificial feeding of infants have been
due chiefly to the non-observance of this principle. It is obvious that
the mother's milk can never be replaced satisfactorily by some other
milk, or milk-substitute. This accounts for the greater mortality among
" bottle babies " than among those that are breast-fed. It is our duty
to make it generally known that on the one hand there is no perfect sub-
stitute for the mother's milk, and on the other hand to show that when
a replacement is unavoidable, the food should be adjusted in accordance
with the requirements established as a result of biological investigation.

Chlorine is also an important constituent of milk. It occurs as chloride
of sodium and of potassium, and is distributed throughout all the cells of
the body. The alkali chlorides, especially the sodium compound, play
an important part in the formation of the urine. We shall come back
again to the relations of chlorides to the juices of the stomach.

Finally, milk contains another element, sulphur, which is present in
a firm state of combination in the proteins casein, albumin, and globulin.
In this connection the reader is referred to what was said concerning the
decomposition products of protein.'

As far as we know, this comprises all the elements contained in milk.
It is, to be sure, possible that other elements are present in small amounts.
Thus, it has been suggested that milk may contain iodine. This assump-
tion was made merely because iodine plays an important part in the
economy of the cells. It is, however, perfectly possible that the new-
born child either contains iodine already stored away, or else that it makes
use of what it has only for later functions.

There has been a great deal of discussion as to whether the animal
organism normally contains arsenic. It is certain, however, that if such
be the case, the amount present is extremely small. The question con-^
cerning the arsenic content is an old one, and has been zealously discussed
by toxicologists in medical jurisprudence.^ The contradictory results
concerning the normal occurrence of arsenic in the thyroid gland is prob-

' Cf. Lecture VIII, p. 157.

' M. Orfila: Traits de m^decin l(5gal, 4th edition, Vol. Ill, part I, p. 285 (Paris, 1848).

INORGANIC FOODS. 407

ably due to differences in the material examined. The amount of arsenic
in animal tissues must depend upon the nature of the food. Gautier '
and Bertrand- have found arsenic, while others, as, for example, Kunkel,'
have not.

It should be mentioned finally that recently the claim has been made
that lithium is also a normal constituent of the human organism. Erich
Herrmann * found this element present in stages of development where
the nourishment had been provided solely from the blood of the mother.
The lungs were found to be particularly rich in lithium.

From what has been said, it is apparent that inorganic salts are of
great importance as foods, both for the developing and adult organisms.
The cells also require the presence of salts for the proper exercise of their
functions. The cells are constantly being broken down and built up anew.
The more recent investigations concerning the action of the separate salts
make it seem most probable that our knowledge concerning the part that
inorganic substances play in the metabolism of the cells will shortly be
widened greatly, and that before long the inorganic substances in our food
will be the subject of considerable more interest corresponding to their
importance. j

' Compt. rend. 137, 295 (1903), and Bull. soc. chim. Paris, 29, 913 (1903). Cf.
also Compt. rend. 137, 158 and 232 (1903), and Bull. soc. chim. Paris, 29, 639 (1903).

' Ann. inst. Pasteur, 16, 553 (1902); 17, 1 (1903); Ann. chim. phys. 28, 242 (1903);
Bull. soc. chira. Paris, 29, 790 and 920 (1903), and Compt. rend. 137, 266 (1903).

' Z. physiol. Chem. 44, 511 (1905).

• Pfluger's Arch. 109, 26 (1905).

LECTURE XVIII.
OXYGEN.'

All the foodstuffs which we have considered up to this point are intro-
duced into the animal organism by way of the aUmentary canal. There
is one substance required to nourish the body which differs from all the
other organic and inorganic foods, not only in the form in which it is
taken up, but also in the manner of its introduction. We refer to oxygen,
which is taken up as a gas into the animal organism through the lungs
and carried by the blood to the tissues. With the oxygen, as such, there
is no available energy introduced into the organism. It possesses no
chemical kinetic energy, so that it belongs to the same class of substances
in this respect as the salts and water. In every way, however, oxygen
occupies an exceptional position. Plants set it free by the aid of chloro-
phyll and the influence of the sun's rays in the assimilation of carbon
dioxide and water. Energy is required for this process and becomes
stored up as chemical energy. Conversely, in the animal cells oxygen
again combines with the substances formed in the plants, energy is set
free, and we find as the final end-products, water and carbon dio.xide, both
of which can again take part in the cycle.'

The first one to clearly recognize the importance of oxygen for the life
process was Lavoisier. He sharply outlined the important role which
this substance plays in the combustion proce.sses taking place within the
animal organism. With this knowledge, there was laid one of the most
important foundation stones for the entire physiology upon which, in the
period following, stone after stone was piled in rapid succession until
finally the structure was established, the particulars of which we are now
studying. No discovery in the whole field of physiology was so decisive
for further investigation as this. Lavoisier himself, it is true, believed
that the lungs were the seat of all the oxidation processes taking place in
the animal organism. The oxygen taken from the air was supposed to
oxidize the substances brought to the lungs by means of the blood. Such
an assumption was a priori hardly plausible, for in this combustion

' Cf. Christian Bohr: Handbuch der Physiologie der Menschen, Vol. I, p. 54, 1905.

' Fundamentally, there is no such sharp distinction between plant and animal cells.
Plants also utilize chemical energy, but in them the reduction processes far exceed those
of oxidation in the daytime, though in the absence of light (night-time) the latter pro-
cesses are more prominent.

408

OXYGEN. 409

energy is set free which is required by the life-processes taking place in the
tissues and cells. If all combustion took place in the lungs, then at a
single place the greater part of the total energy would be set free. The
tissues and cells could only secure for themselves a part of this energy by
means of certain cleavage-processes. An examination of the gases in the
blood would necessarily decide this question. If the oxygen actually
combined with the combustible substances directly in the lung.s, then it
was certainly to be expected that the blood itself would contain but
little if any oxygen. This idea appealed to Magnus/ who analyzed the
gases in blood and showed that a certain amount of oxygen was present
until the capillaries were reached and at this point a part of it began to
disappear. This proved beyond question that all of the combustion
processes could not take place in the lungs. It left undecided the
question whether the oxidation processes took place e.xclusively in the
blood, or whether oxygen passed through the walls of the blood-vessels
into the tissues. It is conceivable that the tissues constantly give up
these oxidizable substances to the blood. In fact, certain discoveries
support this a.ssumption. If the supply of oxygen be entirely cut off
from an animal, it suffocates. Its blood then contains but traces of
oxygen. On exposing such blood to oxygen, the latter disappears in a
short time, and the amount of carbon dioxide in the blood increases.
The blood of an animal which has not been suffocated shows the same
phenomenon, but to a much less extent. Ludwig and Schmidt,^ who
carried out these experiments, explained this discovery on the assumption
that oxidizable substances were constantly being given up to the blood
which immediately underwent combustion provided the supply of oxygen
were adequate. If this supply were cut off, these substances began to
accumulate in the blood. Now we know that the blood contains cells,
the white and red blood corpuscles, which themselves undergo metabolism,
and thereby may easily consume oxygen and yield carbon dioxide. The
above experiments, therefore, are not sufficient to prove satisfactorily
that the combustion takes place chiefly in the blood. Afonassiew ^ then
showed that, as a matter of fact, only the blood-corpuscles and not the
serum of a suffocated animal could take up oxygen in this way. The
assumption that the combustion takes place in the cells and tLssues them-
selves was furthermore supported by the following experiment: Pfluger
and Oertmann * removed the blood from a frog, washed out the last
blood corpuscles with a 0.75 per cent solution of common salt, and finally

' Ann. Physik. 40, 583 (1837); and 64, 177 (1845).

^ Ber. iiber die Verhandl. der Sachs. Ges. Wissen. Leipzig. Math.-physikal KJasse,
19, 99 (1867).

' Ibid. 24, 253 (1872).

' E. Oertmann: Pfliiger's Arch. 15, 382 (1877); E. Pfliiger: ibid. 10, 251 (1875).

410 LECTURE XVIII.

replaced all the blood in the various vessels by this saline solution.
This animal was then placed in an atmosphere of pure oxygen, and con-
sumed as much of the gas and evolved as much carbonic acid gas as a
normal frog.

To-day there is not the slightest possible doubt that oxygen diffuses
into the tissues, and that the cells themselves obtain their energy by the
combustion of their nutriment, which takes place in their immediate
vicinity. We know a great many facts which are in harmony only
with this assumption. One of the principal proofs is that the blood
itself possesses no oxidizing properties.' If, for example, salts of lactic
acid, acetic acid, etc., are placed in the blood they remain unchanged,
whereas in their passage through the organism they are quickly and
completely oxidized. This experiment becomes more convincing if
carried out with surviving organs. If, for example, blood is conducted
through the liver of a dead animal by the portal vein, it can be shown that
ammonium formate introduced into the blood disappears and in its place
urea is formed. This is never the case, however, if the formate is merely
exposed to the action of the blood without coming in contact with the
liver-cells; in such cases the ammonium formate remains unchanged.
Evidently there is a mutual action between the blood and the cells of the
liver which is necessary to cause this complicated reaction to take place.
This is merely one example out of many.

The fact that oxygen actually passes through the walls of the blood-
vessels is strikingly shown by the way the foetus is provided with this
element. It is well known that there is no direct connection between
the vascular system of the mother and that of the child. The circu-
lation of the foetus is isolated. The umbilical arteries carry the blood
rich in. carbon dioxide and poor in oxygen from the foetus through the
umbilical cord to the placenta. Here these arteries break down into
extremely fine branches. They change into the form of chorionic villi in
the enlarged capillaries of the mucous membrane, in the intravillous
spaces of the decidua. To this region the organism of the mother sends
blood rich in oxygen. In order that this oxygen may enter the foetal
circulation — i.e., that it may enter into the umbilical veins, — it must first
penetrate the epithelium and vascular walls of the chorionic villi, and
conversely the venous foetal blood of the umbilical arteries gives up its
carbon dioxide in the same way.

Another proof that the oxygen passes through the walls of the blood
capillaries lies in the fact that the saliva contains a constant amount of
free oxygen. According to E. Pfliiger,^ it contains 0.5 per cent by volume

' Cf. E. Pfluger: Pfluger's Arch. 6, 43 (1872); Hoppe-Seyler:.i6id. 7, 407 (1873).
' Pfluger's Arch. 1, 686 (1868). See also R. Kulz: Z. Biol. 23, 321 (1887). J. L.
Bancroft: Biochem. J. 1, 1 (1906).

OXYGEN. 411

of this gas. This oxygen must come from the'circulation, and is undoubt-
edly to be regarded as oxygen that has not been consumed by the oxida-
tion processes taking place in the cells of the salivary glands.

Paul Ehrlich ' has proposed a very pretty method for following the
course of the oxidation processes taking place in the tissues. If a dye-
stuff which becomes decolorized on reduction and again resumes its color
on oxidation is injected into the veins of an animal, it is easy to recognize
the presence of oxidizable substances in the tissues. Methylene blue is
especially suited for such experiments. If this has been injected into the
veins, it will be found that a freshly- killed animal will be of normal color;
but after being exposed to the air for some time, the color of methylene
blue eventually appears, showing that the tissues have contained this
dyestuff in a reduced form.

The assumption that the consumption of oxygen must actually take
place in the tissues and cells has been based frequently upon numerous
observations concerning the oxygen supply of lower organisms. Thus
the observations of Kupffer ^ and of JMax Schultze ' regarding the direct
supply of oxygen to the cells of the body are a good example. The former
showed that insects which had no real vascular system conduct the atmos-
pheric oxygen directly to the tissues by means of an infinitely-fine tracheal
system. The finest little runners of the tracheae send out branches to the
individual cells, so that the latter by means of these tiny tubes take the
oxygen directly from the air. Again, Schultze showed that in the organs
of phosphorescence of Lampyris splendidula, the finest ends of the tracheae
lead to the individual cells, which cause the phosphorescence.

Although these observations undoubtedly indicate the ability of highly
organized animals to take up and<utilize directly the oxygen of the air,
yet they do not prove conclusively that also in the highest organized
animals such a direct introduction of the oxygen to the cells actually
takes place. In the ascending series of animal species, with the division
of labor and specialization of the separate cell groups becoming more and
more complicated, it would not seem impossible that perhaps one par-
ticular cell group may be limited to quite restricted functions; and that,
for example, the cells receive their energy, in the more highly developed
organisms, from certain cleavage processes, while the energy produced
by oxidation serves merely as the source of heat for the organism. We
have already seen * that intestinal parasites, and even frogs, can live for

' Med. Zentrb. 1886, 113. Das Sauerstoffbediirfms des Organismus, Berlin, 1886.
Cf. C. A. Herter: Z. physiol. Chem. 42, 493 (1904), and .\m. J. Physiol. 12, 128 (1904).
Herter and Richards: ibul. 12, 207 (1904). C. A. Herter: ibid. 12, 457 (1905).

' Beitriige zur Anatomie und Physiologie (1875). Cf. E. Pfliiger: PflUger's Arch.
10, 251 and 270 (1875).

' Arch, mikros. Anat. 1, 124; 6, 186.

* Lecture IV, p. 74.

412 LECTURE XVIII.

a long time without oxygen, and produce carbon dioxide; and, on the other
' hand, we have cited the experiments of Fick and Wislicenus,' who showed
that the energy set free in the cleavage processes was altogether insuffi-
cient to account for the work which these authors were capable of
accomplishing. We came to the conclusion then, that under some circum-
stances the muscular cells, in order to satisfy the demands placed upon
them, must utilize all the chemical energy available from the food
materials it receives.

On the other hand, the assumption that the cells in general are satisfied
to accomplish their work with the energy resulting from cleavage processes,
is supported by the fact that there are unicellular organisms which not
only do not require oxygen, but on which in fact this gas even acts as a
poison. These are the anaerobic bacteria. All sorts of varieties of bacteria
are known, ranging from those to which oxygen is indispensable to those
which get along best without it. There are, in fact, bacteria which are
temporarily anaerobic; i.e., they can get along without oxygen for a time.
It is characteristic of all these bacteria that they eliminate carbon
dioxide, no matter whether they take up oxygen directly from the air or
not. The bacteria which are wholly anaerobic, and those which are tem-
porarily so, must be able to obtain oxygen from the nutriment upon which
they subsist. In the latter case, the assumption might be made that they
store up compounds rich in oxygen, which they consume during the anaero-
bic period, just as the muscular cells are evidently capable of storing up
oxygen, when they are in a state of rest, which they require when the
muscles are being used.

We shall later on - go more into detail concerning the significance of
this progressive breaking down by stages of the nutriment on the part of
the cells in our body, and shall find that by means of this alternate simple
decomposition and oxidation it is possible to obtain energy from the food
as it is required. At all events, all our observations indicate that each
individual cell in the body must have the possibility of obtaining oxj'gen
for oxidation processes, and for the regulation of its internal economy.
We shall soon become acquainted with facts which compel us to accept
this assumption.

Let us now attempt to trace the course of the oxygen from the time it
is taken up by the lungs till it is given up to the cells of the individual
tissues. The blood plays an intermediate part in the process. It takes
the oxygen from the lungs and gives it up to the tissues. The first gas-
exchange is commonly spoken of as external respiration, and the latter as
internal respiration. The question that interests us first of all is how does
the oxygen circulate in the blood. There are two possibilities to be con-

' Lecture IV, p. 69.
' See Lecture XIX.

OXYGEN. 413

sidered. The oxygen may be simply absorbed by the blood, or it may be
that there is some compound in the blood which unites with this oxygen.
In the former case the absorption of oxygen must follow the gas laws/ of
which we shall briefly sketch the most important particulars.

The absorption of a gas l\y a liquid, when there is no chemical reaction
between the two, is dependent upon the nature, the temperature, and the
pressure of the gas; and, in fact, the tveiyht of gas which is absorbed by a
definite liquid is proportional to the pressure under which the gas is placed.
Now according to Boyle's law,- the iveight of a definite volume of a gas is
directly proportional to the pressure, so that evidently the volume of gas
absorbed is independent of the pressure. Again, if instead of a single gas
a mixture of gases stands above a liquid, then each individual gas will be
absorbed independently of the others, and the amount absorbed is gov-
erned entirely by the pressure which this gas exerts (Dalton's law).
This pressure is called the partial pressure. The partial pressure can be
computed as soon as one knows the total pressure exerted by all of the
gases present in the mixture, and the percentage composition of the mix-
ture. The partial pressure is the same percentage of the total pre.ssure
that the gas in question is present in per cent by volume in the mixture.^

If a liquid is allowed to remain in contact with a definite gas mixture
for some time, the liquid will become saturated with gas. When this
has taken place, then the pressure exerted by each gas in the liquid is
equal to the partial pressure of the same gas in the mixture above the
liquid. There is a state of equilibrium between the gas in the atmosphere
and that in the liquid. If this equilibrium is disturbed, for example, by
diminishing the amount of gas in question in the gaseous mixture, then
the liquid will give up this gas until once more the pressure in the mixture
is in equilibrium with the pressure exerted by the gas in the liquid. This
fact may be taken advantage of, if we wish to determine in a simple
manner what pressure is exerted by a gas which is dissolved in a liquid.
The liquid is placed in contact with a gas mixture of a definitely known
composition and pressure (whereby, as stated above, the partial pressure

' Cf. Text-books on Physics. For comparative purposes the volumes of gases are
reduced to 0° C. and 760 miUimeters, barometric pressure. .\s regards the methods
used for examining the gases in blood, see E. Pfiiiger's Untersuchungen aus dem physi-
ologischen Laboratorium zu Bonn, Berlin, 1865. Alexander Schmidt: Verhandl. Sachsi-
Bchen Gcsellsch. Wissensch. 19, .30 (1867). A. Kossel and A. Raps: Z. Physiol. C'hem.
17, 644 (1893). Neesen: ibid. 22, 478 (1897). Midler: Pfluger's Arch. 103, 541 (1904).
J. Geppert: Die Gas-analyse und ihre physiologische Anwendung nach verbesserten
Methoden, Berlin, 1886.

^ The name Mariotte's law is often given to this principle (earlier discovered by
Boyle), that at any given temperature the volume of a given weight of gas varies
inversely as the pressure which it bears.

' The partial pre.ssure of a gas in a mixture is the same pressure that the gas exerts
when present by itself in the volume occupied by the mixture.

414 LECTURE XVIII.

exerted by the gas in question may be accurately computed), and the liquid
shaken with this gas mixture for some time. Now, according as the gas
contained in the liquid exerts a less pressure, the same pressure, or a greater
pressure, than is exerted by the same gas in the gas mixture, there will be
either an increase, no change, or a diminution in the amount of the par-
ticular gas above the liquid.

Let us now come back to the question of the condition of the oxygen
as it circulates in the blood. According to the above principles, it ought
to be easy to decide whether there is any chemical combination between
the oxygen and the blood. From the amount of oxygen in the air that is
breathed into the lungs, or, what is the same thing, from the partial pressure
of the oxygen in the alveolar air, taking into consideration the temperature
of the body and the composition of the blood, we can compute how much
oxygen the latter could take up by simple absorption.

There are a number of different methods which are derived from the
above principles for determining the gas content of a liquid. Since with
rise of temperature the amount of gas absorbed diminishes, it is evident
that the gas can be expelled from a liquid by heating it. When the liquid
begins to boil, i.e., when it is itself being converted into vapor, all of the
absorbed gases have been expelled. The pressure of the gas in the liquid
is now equal to zero, whether it is due to the fact that there is a complete
vacuum above the liquid, or because the gas which was absorbed by the
liquid has been replaced by some other gas (e.g., water vapor) in the
atmosphere directly in contact with the liquid. According to the above
description of the behavior of gases, the effect in the latter case, as far as
the absorption of the gas goes, must be exactly the same as if all the gases
had been removed, for then the partial pressure of the given gas in contact
with the liquid would have become zero.

The air that is breathed into the lungs contains in round numbers 79
per cent by volume of nitrogen, 21 per cent oxygen, 0.03 per cent of carbon
dioxide, and varying amounts of water vapor. If we compare the amount
of oxygen taken up by the blood in the lungs, with that computed to be
present from the partial pressure of the oxygen in the gas mixture that
reaches them, it is found that far too much oxygen is removed by the
blood, so that it is quite out of the question to consider the phenomenon
as one of simple absorption." Nitrogen and argon, which, as far as we
know, take no part in the metabolism of the living animal organism, behave
quite differently when in contact with the blood. They are, for the most
part, merely absorbed mechanically. The absorption coefficient for nitro-
gen amounts at the body temperature to about 0.013 to 0.02. Oxygen,
however, in its absorption by the blood, in no way follows the gas laws.
The amounts of oxygen absorbed by the blood, when it is exposed to differ-

' Cf. Hufner: Arch. Anat. Physiol. 1890, 1; 1895, 209.

OXYGEN. 415

ent partial pressures of this gas, show, within certain limits, but a slight
variation. The blood-plasma is able to take up only 0.65 per cent by-
volume of oxygen.' As a matter of fact, the arterial blood contains more
than 70 times as much oxygen. Again, if blood be placed under an air-
pump, the oxygen is not removed from it at all in proportion to the
change in gas pressure. The oxygen does not leave the blood to any
extent until the pressure has been reduced to 358 millimeters Hg.

Since the blood-plasma contains, as stated above, 0.65 per cent by
volume of absorbed oxygen, the rest of the oxygen contained in blood
must be held in some sort of chemical combination; and, obviously, this
union is effected with the blood corpuscles. In the arterial blood of a dog,
Pfliiger^ found, on an average, 22 per cent of oxygen by volume. If now a
solution of hemogloliin is employed, containing the same amount of hemo-
globin as the blood, it will be found that this solution, within narrow limits,
is capable of absorbing the same amount of oxygen as the blood. This
shows that it must be the hemoglobin which combines with the greater
part of the circulating oxygen in the blood. When hemoglobin
absorbs oxygen, it is changed into oxyhemoglobin; one molecule of
hemoglobin unites with one molecule of oxygen, or for one gram of hemo-
globin there are required 1.56 cubic centimeters of gas (measured at 0° C.
and 760 millimeters pressure). Dog's blood contains approximately 14.5
per cent of hemoglobin. From this it is evident that 22.6 per cent by
volume (1.56 X 14.5) of oxygen can be absorbed by the blood of a dog.
This agrees well with the amount found by actual experiment. It is, how-
ever, not permissible to apply the results obtained by working with hemo-
globin solutions directly to the absorption of oxygen by the blood under
different conditions. There are a number of facts known which show
that there are certain differences as regards the behavior of the two liquids.
This may be accounted for in different ways. It is conceivable that certain
changes may have taken place in the preparation of the hemoglobin solu-
tion. On the other hand, the possibility exists that the hemoglobin in the
blood is influenced by the way it is contained in the blood corpuscles. It is,

' Christian Bohr, Skand. Arch. Physiol. 17, 104 (1905), has recently computed the
absorption coefficients of the plasma and blood for different gases:

Oxygen.

Nitrogen.

Carbon Dioxide.

Water

15°C. 38°C.
0.0342 0.0257
0 033 0 023
0.031 0 022
0.028 0.019

15°C. 38°C.
0 0179 0.0122
0.017 0.012
0.016 0.011
0.014 0.009

15° C. 38° C.
1.019 0.555

Plasma

Blood

Blood corpuscles ....

0.994 0.541
0.937 0.511
0.825 0.450

E Pflijger: Zentrb. med. Wiss. 1867, 722, and Pfluger's Arch. 1, 274, 288 (1868).

416 LECTURE XVIII.

moreover, very questionable whether hemoglobin itself is a simple substance.
The component containing iron is constant in its composition, but the
relation between this and the globin (the protein constituent), or, in other
words, the numberof globin molecules which unite with the hemochromogen,
varies in different cases. It is necessary to mention this fact in this con-
nection, because it is unquestionably true that differences in the results
obtained by investigators are due, to some extent at least, to the fact that
the results obtained by working with hemoglobin solutions have been
applied directly to the absorption by the blood.

Hemoglobin itself consists of a protein, globin, and another constituent,
hemochromogen,' which contains iron; it is the hemochromogen alone that
unites with oxygen. This is shown by the fact that hemochromogen absorbs
as much oxygen from the air as an equivalent amount of hemoglobin.
When hemochromogen is oxidized to hematin, the hemoglobin of the blood
becomes oxyhemoglobin. Whereas the oxygen combined in hematin can-
not be removed by means of an air-pump, this is not the case with oxy-
hemoglobin itself. This fact is of great importance for the understanding
of the transportation of oxygen by the blood and its giving up of the same
to the tissues. Oxyhemoglobin belongs to the class of compounds which
are said to be dissociable. Before we go into further particulars concern-
ing this transportation of oxygen by the blood and the subject of internal
respiration, we must make perfectly clear what are the conditions in the
animal organism upon which the dissociation of the oxyhemoglobin de-
pends. We have already mentioned the fact that the blood-plasma contains
absorbed oxygen. The amount present is, corresponding to the gas laws,
relatively small. It is perfectly clear that it follows the laws of gas absorp-
tion. First of all, the amount of this gas must be in equilibrium with the
alveolar air. On the other hand, this absorbed oxygen, in its transporta-
tion to the various tissues, must necessarily constantly seek to be in
equilibrium with the pressure of the oxygen in these tissues, and, hkewise,
in accordance with the well-defined gas laws. Now, as we shall soon see,
the tissues are constantly consuming oxygen and forming carbon dioxide
therefrom. For this reason the pressure of oxygen in the tissues is kept
lower than that of the absorbed (or dissolved) oxygen in the blood. Hence
oxygen is constantly entering these tissues from the blood. There is no
doubt at all that, in the first place, this absorbed o.xygen is given up. Now
in proportion as this absorbed oxygen is given up by the blood, oxyhemo-
globin becomes dissociated, i.e., begins to give up its oxygen to the plasma.
If this conception be correct, it must be possible to remove eventually
all the oxygen from the blood by means of the air-pump, even at
low temperatures, i.e., without the aid of heat, which itself tends

■ Cf. Lecture VII, p. 141.

OXYGEN. 417

to dissociate the oxyhemoglobin. This has, in fact, been found to
be the case.' For the cells themselves, therefore, at any given
moment, only the uncombined oxygen is available. The oxygen con-
tained in the oxyhemoglobin serves, as it were, as a reserve supply. It
is only the merely mechanically absorbed oxygen which determines the
pressure of the oxygen in the blood, and determines therebj^ the gas
exchange. Naturally the extent of the giving up of oxygen to the tissues
depends solely upon the magnitude of this pressure exerted by the absorbed
oxygen in the blood. The fact that the oxygen in the blood is not merely
absorbed, but for the most part held in a state of loose combination, is of
great significance for the entire metabolism. The animal organism is,
within fairly wide limits, independent of the partial prefssure of the oxygen
in the surrounding atmosphere. From rarefied air, the blood removes
the oxygen and combines it with hemoglobin. We can imagine that the
process takes place in somewhat the following manner: First of all the
oxygen is absorbed by the blood-plasma from the alveolar air in accord-
ance with the gas laws; but inasmuch as this dissolved oxygen constantly
tends to combine with the hemoglobin, more and more oxygen is taken up
from the air. In this way a considerable store of oxygen is laid away in
the organism, by means of which it is able to satisfy, at any time, any
unusual and unexpected demands for this important gas.

The question next arises as to how the amount of oxygen that is taken
up by arterial blood corresponds to the quantity absorbed when a sample
of blood is thoroughly shaken with air. Experiment has shown that
normally the blood is nearly saturated with oxygen, for but little more is
absorbed when it is shaken with air. The taking up of oxygen by the
blood is dependent upon certain definite conditions. This is evidently true
of a small amount that is mechanically absorbed by the plasma. The
amount of oxygen which unites with the hemoglobin, on the other hand,
is entirely independent of the laws which govern the absorption of gases
in cases where no chemical combination takes place. Paul Bert ^ studied
the influence of the temperature. At higher pressures he was not able to
detect an}' definite influence, but with lower gas pressures there was less
absorption at the temperature of the body than at, say, the room tempera-
ture. The fact that the absorption of the oxygen depends upon the pres-
sure of the gas is clearly shown by the following table prepared by Krogh.'
Krogh examined the blood from horses at 38° C, and determined the
amount of chemically combined oxygen; i.e., from the total amount of

' Christian Bohr: Blutgase und respiratorischer Gaswechsel, Handbuch der Physio-
logie des Menschen. Vol. I, pp. 221, 222 (1905).

' La pression barom^trique, Paris, 687 (1878). Cf. also A. Lowy: Zentr. Physiol. 13,
449 (1899); Arch. Physiol. Anat. 1904, 231 and 565.

' Skand. Arch. Physiol. 16, 390 (1894).

418

LECTURE XVIII.

oxygen absorbed, the small amount merely absorbed by the plasma was
deducted. These values may be represented graphically by plotting the
oxygen pressures as abscissa, and the amount of oxygen absorbed as
ordinates. Two such plotted curves representing the curves of oxygen
tension, as Bohr called them, are shown below in which the dotted line
represents the absorption by the plasma, the other that quantity which
is chemically bound.

Horse Blood at 38° C.

Pressure of Oxygen in

In 100 c.c. Blood.

Oxygen Absorbed.

Millimeters.

Combined Oxygen.

Oxygen Dissolved

Per cent Chemi-

Dissolved in 100

in the Plasma.

cally Combined.

c.c. Plasma.

10

6.0

0 020

30.0

0 030

20

12 9

0.041

64.7

0.061

30

16.3

0.061

81.6

0.091

40

18.1

0.081

90 4

0.121

50

19.1

0.101

95.4

0.152

60

19.5

0.121

97.6

0.182

70

19.8

0.141

98.8

0.212

80

19.9

0.162

99.5

0.243

90

19.95

0.182

99.8

0.273

150

20.0

0.303

100

0.455

In the above table column 4 shows the per cent of chemically combined
oxygen, placing arbitrarily that absorbed at 150 millimeters pressure at
100 per cent.

— ^=-— 1 — I — I —

10 10 30 40 iO 60 70 80 90 100 110 120 130 140 130

Fig. 3. Absorption of Oxygen at Various Pressures by Horse's Blood at 38°.

These two curves show very clearly the different behavior of the chemi-
cally combined oxygen, and that which is nearly absorbed. The amount
of the latter increases regularly in proportion to the pressure as it should

OXYGEN. 419

in accordance with the gas laws. The chemically bound oxygen, on the
other hand, is only affected materially by low pressures.

We must not fail to mention that recently Ch. Bohr ' has made certain
observations which tend to shatter the belief that the oxygen combination
by hemoglobin is a constant quantity." Bohr speaks of a specific oxygen
capacity of the blood in different animals and different individuals. The
proportion of hemoglobin, or of the iron contained in it, to the amount of
oxygen consumed, varies. In order to explain this fact, Bohr makes the
assumption that the pigment in the blood is not a simple substance, but
is composed of different components, which separately combine with vary-
ing amounts of oxygen. It is conceivable that each individual blood
corpuscle originally incloses a uniform pigment, the specific nature of
which is gradually attained in various ways. This would account for
the fact that iron and oxygen are often found in unequal atomic propor-
tions. It must be emphasized, however, that Bohr's assumption is to be
regarded merely as an hypothesis. It is by no means satisfactorily proved.
We have mentioned these investigations of Bohr, partly because they open
up again to experimental research one of the few fields which had appar-
ently been investigated exhaustively. Once more our interest is aroused
concerning all the questions regarding the transportation of oxygen,
new inquiries are suggested, and a process which has been regarded as
simple, is perhaps to be looked upon as of quite complicated mechanism.

In order to understand correctly the transportation of oxygen in the
blood, the process by which it is taken up in the lungs and given up to
the tissues, we must for the present stop attempting to trace the course
of the oxygen, but concern ourselves with one of the end-products which
results from the oxidation (combustion) of the organic substances in the
tissues; namely, carbon dioxide. This is necessary, because, according to
Bohr's investigations, it is evident that definite relations exist between the
oxygen content of the red corpuscles and the carbon dioxide content of
the blood. The carbon dioxide in the blood comes, as we have said, from
the tissues. It is formed everywhere in the cells as one of the end-products
in the metabolism of oxygen. There must, therefore, be developed in the
separate tissues a certain carbon dio.xide gas pressure, which is in equilibrium
with the pressure exerted by this gas in the surrounding cell-complex, and
also with that in the blood. When the arterial blood, freshly laden with
oxygen, passes through these tissues, which are rich in carbon dioxide,
then gas will diffuse into the blood, for the pressure exerted by the carbon
dioxide in the blood is less than that of the tissues. The amount of carbon
dioxide in arterial blood has been found to average 40 per cent by volume,
but this varies greatly. In venous blood, i.e., that which is flowing away

' Handbuch der Physiol, loc. cil. p. 93.

' G. Hufner: Arch. Anat. Physiol. 1894, 130.

420

LECTURE XVIII.

from the tissues, there is more of this gas. The following table will give
some idea of the amounts of the separate gases in arterial and venous
blood.'

Oxygen.

Carbon Dioxide.

Nitrogen.

20
12

43.6
50.0

1.2

1.2

The venous blood from different vascular regions shows an extremely
varying content of the different gases. The carbon dioxide content of all
the venous blood taken as a whole, must be represented by that of the
right heart; for it is here that the venous blood coming from the whole
vascular system is mixed. Schoffer,^ working in Ludwig's laboratory in
1860, compared the composition of arterial blood with that of the right
heart. The following table gives the results obtained by Bohr and
Henriques.

Oxygen.

Carbon Dioxide.

Nitrogen.

Artery.

Riglit Heart.

Artery.

Right Heart.

Artery.

Right
Heart.

I. . .
-II. . .
III. . .

25.6
21.3
20.3

17.3
11 9
14.4

44.0
42.6
45.9

51.5
48.5
50.3

1.23
1.19
1.18

1.31
1.06
1.40

Average

22.4

14.5

44.2

50.1

1.20

1.26

If we compare the amount of carbon dioxide present in arterial blood
with that in venous blood, it is at once apparent that it cannot be a case of
simple absorption. The quantity present is far too large. Like oxygen
it must be chemically combined for the most part. The amount of carbon
dioxide absorbed by the blood does in fact depend upon the pressure of the
gas, with which it is in equilibrium; but the absorption is not proportional
to this pressure, as it would be in a case of simple absorption. With what
substance in the blood is this gas combined? The relations here are far
more complicated than in the case of oxygen. With the latter there ia
but one kind of combination, — that with hemoglobin. Carbon dioxide,
however, combines with different substances in the blood. A part of

' Christian Bohr: Handbuch, loc. cit. p. 83.

» Sitzungsberichte d. Wiener Akad. 41, 613 (1860). C. Ludwig: Mediz. Jahrbucher,
Wien, 1865. N. Zuntz and Hagemann: Ergiinzungsband III zu der landwirtschaftl.
Jahrb. 27 (1898).

OXYGEN.

421

this gas is merely dissolved both by the plasma and by the red corpuscles,
following the laws of gas absorption. The average gas pressure of the
carbon dioxide in the organism may be taken as 30 millimeters. Cor-
responding to this pressure, the amount of carbon dioxide physically
dissolved in 100 cubic centimeters of blood amounts to 2.01 cubic centi-
meters. Now the total amount of carbon dioxide absorbed under these
conditions is about 40 per cent by volume, so that approximately only
5 per cent of the total carbon dioxide absorbed is merely held in solution.

It is of interest to know how the carbon dioxide is distributed between
the blood-corpuscles and the plasma. According to Setschenow,' about
two-thirds of the carbon dioxide in dog's blood is held by the plasma, and
one-third by the blood corpuscles. Kraus - found similar values with the
blood of oxen. In blood from horses, however, Fredericq ^ found only
one-fourth with the corpuscles and three-fourths with the plasma.

A. Jaquet ■* studied the influence of the carbon dioxide gas pressure upon
the absorption of the gas by the plasma at 37.5° C. The absolute values
of this absorption vary greatly. They depend upon the alkalinity of the
plasma. Although the values given in the following table are, therefore,
only relatively true, still they show how the amount absorbed depends
upon the pressure exerted by the gas.

CARBON DIOXIDE ABSORPTION BY THE SERUM.

Pressure of CO2
in mm.

CO2 chemically com-
bined in 100 cc.

Pressure of CO2

CO2 chemically com-
bined in 100 cc.

14.8
16.5
17.0

45.8
57.4
58.5

26.6
42.7

61.7
63.7

It is evident from the above figures that with low pressures there is a
marked increase in the absorption with increasing pressure. With pres-
sures above 20 millimeters, however, this increase is not so marked.

Let us now see what the nature of the chemical combination is between
the carbon dioxide and the plasma. First to be considered are the salts
of the alkalies, especially carbonates. The amount of these present in the
plasma is quite large, the sodium salts predominating. Now we know that
monocarbonates are changed to bicarbonates by the absorption of carbon
dioxide, and conversely that loss of the same gas gives rise to the form-
ation of monocarbonates again. It would thus be very easy to account
for the transportation of the carbon dioxide by the blood. In reality.

' M^moires de I'Acad. de St. Petersburg, 26, 59 (1879).

' Festschrift Graz. p. 19 (1898).

' Compt. rend. 84, 661 (1877); 85, 48 (1878).

* Arch, exper. Path. Pharm. 30, 311 (1892).

422 LECTURE XVIII.

however, the relations are by no means so simple. The dissociation of the
bicarbonate in solutions of the same concentration as the serum at 37° C,
becomes noticeable only when the pressure of the gas upon the solution is
less than a few millimeters. With a pressure of 0.2 millimeter about
three-fifths of the total dissociable carbonic acid still remains chemically
combined. According to the observations of Jaquet, cited above, the car-
bonic acid of the plasma behaves quite differently. Complete saturation
is not effected with 15 millimeters of carbon dioxide pressure. The behavior
of the carbonic acid loosely combined in the plasma, therefore, cannot be
explained by its relations to bicarbonates and monocarbonates. The fact
that by means of the air-pump more than half of this carbonic acid may
be expelled from the plasma, speaks, more than anything else, against any
such assumption. Inasmuch as we know of no other compounds in the
plasma which would be capable of uniting with carbon dioxide to any
considerable extent, we are forced to believe that other weak acids are
present in the plasma which are constantly striving to unite with the
alkali. Sertoli ' long ago looked for such acids, and considered as such
the protein substances of the plasma, especially the globulins. To-day
there is no longer any doubt that these protein substances are actually
present in the form of alkali salts in serum. They are driven out of these
compounds if there is an excess of carbonic acid present. N. Zuntz and
A. Lowy- have shown this assumption to be true in a very convincing
manner. They found that the amount of diffusible alkali in the serum
increased by conducting carbon dioxide into it. This is to be attributed
to the fact that as the carbonic acid is forced into the serum, the alkali
albuminates which are not diffusible are decomposed, and alkali carbonates
which are capable of passing through the membrane are formed in their
place.

The next point to be decided is whether the alkali contained in the
serum is entirely combined with albumin when the partial pressure of the
carbon dioxide gas is equal to 0, or whether an excess of alkali is present ?
If, other than alkali albuminates, there were no other alkali salts of weak
acids present in the serum, then it would be expected that if the alkali
were completely combined with the protein (i.e., when the partial pressure
of the carbonic acid gas was zero), all of the carbon dioxide would
have been driven out of the plasma. This is not the case, as E. Pfluger ' has
shown. In one experiment he found 4.9 per cent by volume, and in
another 9 . 3 per cent of carbon dioxide which remained in the plasma, and

' Sertoli: Hoppe-Seyler, Medizin-chem. Untersuch. Berlin, 1868, p. .350. Cf. N.
Zuntz: Hermann's Handbuch der Physiol. Bd. 4, 64 (1882). Torup: Die Kohlensiiure-
spannung des Blutes, Kopenhagen, p. 36 (1887). Kurt Brandenburg: Z. klin. Med. 45,
H. 3 and 4.

■' Pfluger's Arch. 58, 511 (1894).

^ Die Kohlensaure des Blutes, p. 11, Bonn. 1864.

OXYGEN. 423

could not be removed by the air-pump. Pfliiger proved this amount of
carbonic acid to be present by adding acid to the plasma. In other words,
Pfliiger assisted the action of the protein, which was insufficient to take
the place of all the carbonic acids in the blood, by the artificial addition of
a stronger acid, which expelled the remainder of the carbonic acid that was
combined with the alkali.

From these experimental results, we can describe the combination of
the carbonic acid in the plasma, somewhat as follows : A part of the
carbon dioxide is evidently present, even with low pressures of gas, as
bicarbonate. With increasing carbon dioxide pressures, a part of the gas
replaces the protein in its combinations with the alkali. In this way we
are able to understand much better how the carbon dioxide gas exchange
takes place, although it cannot yet be said that the entire process has been
satisfactorily explained.

The reason that we do not at present understand clearly the exact way
in which the carbon dioxide is combined in the plasma, and have no exact
data concerning the dissociation of the separate compounds, is because
the plasma itself is a complicated mixture of unlike substances, which
mutually influence one another in a number of different ways. The study
of the behavior of the bicarbonates alone, or, on the other hand, of the
albuminates, does not lead to results which can be applied directly to
the plasma, for it is at present impossible for us to imitate precisely
the conditions prevailing in this fluid.

There is no doubt that alkali phosphates which are always present in
the plasma, even although in small amounts, also have an effect upon
the combination with carbonic acid. When exposed to the action of car-
bon dioxide, Na2HP04 is attacked with the formation of NaH2P04 and
NaHCOs.

According to the results obtained bj- Setschenow,' the removal of the
alkali from alkali albuminates takes place only with carbon dioxide pressures
which are greater than those ordinarily prevailing in the living organism.
Thus we may have in these compounds a regulating mechanism which
prevents the carbon dioxide pre.ssures from exceeding a certain maximum.
If the pressure of the gas exceeds this normal value, the alkali albuminates
then serve to unite with the excess of the carbonic acid, and thus prevent
any considerable pressure being exerted by the gas.

Carbonic acid is likewise contained in the blood corpuscles, partly free,
and partly in a state of chemical combination. At 38° C, and 30 milli-
meters gas pressure, there is present in the blood corpuscles corresponding
to 100 cubic centimeters of blood, about 0.6 cubic centimeter of the gas,
which is simply physically dissolved. The greater part of the carbon
dioxide absorbed by the red corpuscles does not follow the laws for gas

» M^moires de I'Acad. de St. Petersburg, 26, GO (1S79).

424 LECTURE XVIII.

absorption. This part is chemically combined, and in fact, chiefly with
the pigment, hemoglobin. This may effect the absorption of carbon
dioxide in two ways. In the first place, the globin in it and the remaining
proteins of the blood, may strive to combine with the alkali, as does the car-
bonic acid. On the other hand, the hemoglobin itself may unite directly
with the carbon dioxide. The first way in which the hemoglobin influences
the absorption of the carbon dioxide is perfectly analogous to what we
have just been discussing with regard to the plasma. Here, also, the union
between the hemoglobin and the alkali will not be dissolved until the pres-
sure of the carbon dioxide has reached a certain value. Thus N. Zuntz '
found the compound between the hemoglobin and alkali was not decom-
posed appreciably, until the pressure of the carbon dioxide was greater
than 70 millimeters. This benefits the organism only in time of excep-
tional need.

We shall now consider the nature of the chemical union between the
carbonic acid and the hemoglobin itself. We have seen that hemoglobin
combines with oxygen, and that this property is peculiar to that part of
the molecule which contains the iron, while the globin participates indi-
rectly in the reaction only in as much as the union of the globin with the
hematin brings forth relations which change the firm state of combination
between oxygen and hematin into one which is more readily dissociable.
It is conceivable that the carbon dioxide unites with the same part of
the hemoglobin molecule that oxygen does. We do in fact know of gases
of which this is true, as, for example, carbonic oxide (carbon monoxide).
One volume of the latter gas replaces one volume of oxygen.^ This car-
bonic oxide compound with hemoglobin is also dissociable. The carbon
monoxide may be replaced by oxygen again. This takes place when the
partial pressure exerted by the oxygen exceeds that of the carbonic oxide.'
The fact that the carbonic oxide actually combines at the same place as
the oxygen was shown to be very probable by Hoppe-Seyler,^ who showed
that it was combined in the iron-containing radicle of the molecule.
Now it is well known that carbonic oxide has a poisonous effect; and appar-
ently this is due to the fact that it replaces the oxygen in its combination
with the hemoglobin, so that it seriously affects the supply of oxygen for
the tissues. Such an action is unknown in the case of carbon dioxide. It
is also a priori hardly probable that oxygen and carbonic acid should each
strive for possession of the hemoglobin molecule. It has also been shown

' Zentr. med. Wiss. 6, 529 (1867).

' G. Hufner: Arch. Anat. Physiol. 1895, 209. Hufner and Kiilz: J. pr. Chem. 28,
256 (1883) ; and Hufner: rhi'fl. 30, 68 (1884).

Recently Hufner and Kiister have undertaken experiments to determine the rela-
tion.s by weight in which heraochromogen combines with carbonic oxide. Arch. Anat.
Physiol. 1904, 387,

* Z. Physiol. Chem. 13, 477 and 493 (1889).

OXYGEN.

425

that the carbon dioxide is taken up quite independently of the union of
hemoglobin with oxygen.

This is made very clear by the experiments of Bohr.' He showed that
the presence of oxygen had no apparent effect upon the amount of carbonic
acid absorbed under different pressures. The hypothesis is also supported
by the fact that the transformation of hemoglobin into methemoglobin'
does not in any way affect its combination with carbon dioxide,' while that
with oxygen is disturbed. It seems probable that the carbon dioxide is
not combined with the hematin part of the molecule, but rather with the
globin. This appears the more probable since M. Siegfried * has recently
shown that carbonic acid is associated (i.e., chemically bound) by the
action of amino acids and protein substances. From such compounds it
is very easy to set the carbonic acid free again; i.e., dissociate it. From
glycocoll, for e.xample, a carbaminoacetic acid is formed. From the
amphoteric amino acid a relatively strong dibasic acid results.* The
carbamino acids correspond to the general type :

H

/
R— N

I ^COOH.
COOH

The union of carbonic acid with globin is also dependent upon the pres-
sure of the gas, especially when this is low. This is shown by the following
table. It gives the amount of carbonic acid chemically combined per gram
of hemoglobin at different gas pressures of carbonic acid. The concentra-
tion of the hemoglobin solutions amounted to 2.69 per cent; the temper-
ature was 38° C.

Tension of COa in

CO2 Absorption per

Tension of CO2 in

CO2 Absorption per

Millimeters.

Gram of Hemoglobin.

Millimeters.

Gram of Hemoglobin.

10

1.260

60

2 363

20

1.647

100

2.701

30

1.902

200

3.113

40

2 091

300

3.312

50

2.240

3.990

' Zentr. Physiol. 4, 49 and 253 (1890); and Skand. .\rch. Physiol. 3, 47 and 64 (1892).

' See Lecture XXIV.

' Skand. Arch. Physiol. 8, 363 (1898).

' Z. Physiol. Chera. 44, 85 (1905) ; 46, 490 (1905). See Lecture XI, p. 2.35.

' It is evident that such compounds may be formed in the tissues, and that in this
way any momentary excess of carbonic acid can be combated. Thus, the cells can
assist the oxidation processes. It is also possible that the carbonic acid assimilation
on the part of plants, as Siegfried has suggested, may also take place through the for-
mation of carbaminic acids, and that the latter, and not the carbonic acid, are reduced.

426

LECTURE XVIII.

The concentration of hemoglobin in the blood amounts to 15 per cent on
an average. Bohr ' computed from the above values that with a carbon
dioxide pressure of 30 millimeters, about 8 . 1 cubic centimeters would be
held in combination in 100 cubic centimeters of blood. Allowing for the
0.6 cubic centimeter of the gas which is held in merely physical solution
by the red corpuscles, then, as the total amount of carbon dioxide absorbed
by the corpuscles at that pressure amounts to about 14 cubic centimeters,
there remains unaccounted for somewhat over 5 cubic centimeters of
carbon dioxide. This must be combined with other substances in the
blood corpuscle. These other substances are evidently the alkalies present
which can form bicarbonates.

We have now considered the absorption of carbon dioxide by the plasma
and by the corpuscles, each acting independently, and it remains to decide
whether such a mixture as is present in the blood has any reciprocal effect
upon such absorption. N. Zuntz - has shown that if an equilibrium has
been established, at a definite carbon dioxide pressure, in the exchange of
the dissociable substances between the plasma and blood corpuscles, that
a change in the pressure of the carbon dioxide will disturb this equilibrium.
Thus an increased pressure of the gas makes the serum more alkaline, while
the chlorine content simultaneously diminishes, as the following table
shows. It demonstrates Ukewise the reversibility of the entire process.^

INFLUENCE OF CARBON DIOXIDE UPON THE COMPOSITION OF THE
BLOOD IN CORPUSCLES AND SERUM.

Specific gravity of serum

Total solids in 50 cc. serum ....

N
Volume of t-: AgNOa solution corre-
sponding to the chlorine in 100 cc
serum

(a) Blood a,
shaken with air.

1 026
4.157

(6) Blood a,
exposed to ac-
tion of CO: for
30 min.

1.030
4.532

(c) Blood b.

after conducting

air through it

for two hours.

1.026
4.122

This increased alkalinity of the serum is explained by assuming a migra-
tion of the alkali carbonates to take place from the corpuscles to the
plasma. There is, however, no experimental evidence in support of this
assumption. Giirber * has shown that the potash is not driven from the

' Handbuch f. Physiol, he. HI. p. 115.

^ Beitrage zur Physiol, des Blutes, Inaug. Diss. Bonn, 1S68.
' H. J. Hamburger: Osmotischer Druck imd lonlehre in den mediz. Wissensch.
p. 263, Weisbaden, 1902.
* Sitzb. physikal. med. Ges. Wurzburg, 1895, 28.

Vol.

OXYGEN.

427

corpuscles by the increased pressure of the carbon dioxide gas. This
author beheves that the carbon dioxide expels the hydrochloric acid from
sodium chloride by virtue of its mass-action. Sodium carbonate is formed,
while the hydrochloric acid, set free, migrates to the blood corpuscles.
The reciprocal relation of the plasma and blood corpuscles has not, how-
ever, been explained satisfactorily up to date.

Then, again, has the carbon dioxide tension of the blood any influence
upon the absorption of oxygen? We have already seen that the converse
is not true, because the carbonic acid and oxygen are combined at different
places in the hemoglobin molecule. It has been believed for a long time
that the pressure exerted by carbon dioxide in the blood does effect the
absorption of oxygen. From the above facts, however, it would seem
hardly probable a priori. Bohr, Hasselbach, and Krogh,* nevertheless, have
shown that as a matter of fact the absorption of oxygen is affected by
carbon dioxide pressures, which are not above the physiological values.
With increasing carbon dioxide pressures the absorption of oxygen becomes
less. A few figures will show how great this effect is:

Oxygen absorbed at CO2 pressures of —

Oxygen pressure

5 mm.

10 mm.

20 mm.

40 mm.

80 mm.

5

11

7.5

5

3

1.5

10

28.5

20.5

14

9

4

15

51

36

27

18.5

8

20

67 5

54

41

29.5

14

40

89

84

77

66.5

49

60

95

93.5

19.5

86

73

80

98

97

96

94.5

87

100

99

98.5

98

97

95

150

100

100

100

99.8

99.5

Bohr explains this by assuming that the entrance of carbonic acid into
the globin molecule changes the aflinity of the globin for hematin, and
thereby influences the absorption of o.xygen by hematin at low oxygen
pressures. The biological significance of these discoveries may be ex-
plained perhaps as follows: We have already seen that, as regards the gas-
exchange with the tissues, it is not the total oxygen contained in the blood
which is effective, but chiefly that which is contained dissolved in the
plasma; it is this that causes the oxygen pressure of the blood. If now
an increased amount of carbon dioxide be produced, then as the hemo-
globin cannot hold as much oxygen in combination as before, there will be
more oxygen in the dissolved state, so that the oxygen pressure of the,

' Zentr. Physiol. 17, 661 (1904), and Skand. Arch. Physiol. 16, 402 (1904).

428 LECTURE XVIII.

blood becomes greater, and there is a more lively gas-exchange between
the tissues and the blood.

We shall now attempt to trace the gas-exchange of the blood with the
alveolar air on the one hand, and with the tissues on the other. In the
lungs, two processes are continually taking place side by side. Blood
laden with carbon dioxide constantly reaches these organs, there to dis-
charge this gas and take up a fresh supply of oxygen. The dark-colored
venous blood is hereby changed into bright-red arterial blood, and this
reenters, through the veins of the lungs, the general circulation. In order
to understand the entire gas-exchange in the lungs, we must remember
that the blood-vessels of the lungs (the region where the pulmonary arteries
end, and the veins begin) represents an infinitely-fine, capillary network,
spun round the alveoli. In this way an enormous amount of surface is
exposed, which enables us to comprehend how, in spite of the relatively
quick passage of the blood through the lungs, a complete gas-exchange
takes place. The size of the respiratory surface has been variously esti-
mated. Aeby ' found that the lung surface in an adult with quiet breath-
ing, amounted to 80 square meters. N. Zuntz,^ assuming the alveolar
diameter of 0.2 millimeter, and the air volume of the lungs to be 3000
cubic centimeters, estimated the alveolar surface to be 90 square
meters.

If we compare, first of all, the expired and inspired air, we shall find that
the former is poor in oxygen and rich in carbon dioxide, in comparison
with the latter. The outer air does not reach the alveoli in an unchanged
condition. It is first saturated with water vapor, and warmed to the
temperature of the body. It originally contains, on an average, 20.96
per cent oxygen, 78 per cent nitrogen, 1 per cent argon, and 0.04 per cent
carbon dioxide by volume. We cannot, however, apply these values
directly to the gas-exchange in the alveoli. For the absorption of oxygen,
on the one hand, and the giving up of carbon dioxide, on the other, it is
the composition of the alveolar air which alone comes into consideration.
The latter is poorer in oxygen, and richer in carbon dioxide, than the
expired air, which contains 16.4 per cent oxygen and 4.1 per cent carbon
dioxide by volume. This is because only a part of the inspired air reaches
the alveoli. A part of it remains unused in the air-passages, where it is
mixed there with the alveolar air and expired. The carbon dioxide and
oxygen content of the alveolar air at the moment it leaves the alveoli
may be computed, if we know the volume of a single inspiration, and the
size of the air-passages which contain the unchanged inspired air (nose,
pharynx, trachje, and bronchi). Such computations, it is true, are not
accurate, partly because this latter value is not known closely enough, and

' Der Bronchialbaum der Saugetiere und der Menschen, p. 90, Leipzig, 1880.
' In Hermann's Handbuch der Physiologie, 4, 90 (1887).

OXYGEN. 429

because the composition of the expired air varies according to the depth
of the inspiration. In this way average values of 14.6 per cent oxygen
and 5.6 carbon dioxide, by volume, have been found for the alveolar air
when it leaves the alveoli. During inspiration, its composition tends to
approach that of the inspired air. Some idea as to the extent of the
changes in the composition of the air in the alveoli, during inspiration, may
be obtained by comparing the amount of inspired air with that remaining
in the lungs at the end of respiration. In ordinary breathing there remain
2800 cubic centimeters of air (1200 cubic centimeters residual and 1600
cubic centimeters of reserve air). As the average inspiration amounts
to only 500 cubic centimeters, of which about 360 cubic centimeters
reach the alveoli, it is evident that the changes in the decomposition of
the alveolar air as a result of inspiration are not very great. The necessity
of knowing the composition of the alveolar air, in each case, has been
realized only as a result of recent investigation. Upon this knowledge
depends our judgment as to whether the gas-exchange in the alveoli,
between the blood and alveolar air, follows simply the laws of gas absorp-
tion, or whether other forces must come into play. It is, to-day, still
believed by many that the first explanation of the gas-exchange in the
lungs is entirely satisfactory. Blood reaches the lungs, through the pulmon-
ary arteries, having a greater carbon dioxide tension than does the alveolar
air. An equilibrium must be established between these two gas-pressures,
and as a result carbon dioxide diffuses from the blood into the alveoli.
Similarly, on account of its greater tension in the alveolar air, oxygen passes
into the blood. The hemoglobin in this way becomes saturated with
oxygen, and is ready once more to enter the general circulation. This
assumption is supported by the work of Wolflberg' and of Nussbaum.^
If, namely, the gas-exchange in the alveoli of the lungs follows exactly
the laws of gas diffusion, then, in a lobule, which is cut off by the
closing of the bronchial tube leading to it, the alveolar air must be
in equilibrium with the carbon dioxide tension of the blood. Similarly
the arterial blood, flowing from this lobule, must have the same carbon
dioxide tension as that of the alveolar air. Wolffberg and Nussbaum
found, as a matter of fact, that the carbon dioxide tension in the alveoli
was the same as that of the venous blood which flows to them. They
introduced a double-walled elastic catheter into a branch of the bronchus
of a tracheotomized dog, in which this portion of the lungs was shut off,
by inflating a rubber enlargement of the catheter. After a short time
a sample of the alveolar air was withdrawn through a tube in the catheter
and its chemical composition determined. They found on an average that
this isolated alveolar air showed a carbon dioxide tension of 3.84 per cent

' Pfliiger's Arch. 4, 465 (1871); 6, 23 (1872)
' Ibid. 7, 296 (1873).

430

LECTURE XVIII.

of an atmosphere. The corresponding vahie for the blood of the right
heart was 3.81 per cent.

According to the results of this experiment, it would seem that we were
unquestionably justified in assuming that the gas-exchange in the alveoli
of the lungs takes place in accordance with the well-known laws of gas-
diffusion. Quite recently, however, and especially by the extended experi-
ments of Bohr,' facts have become known which cannot be explained on
this basis. Bohr desired, in each experiment, to know the composition of
the alveolar air. A good idea of this can be obtained by analyzing the
out-going air, obtained at the moment it passes the bifurcation of the
trachea. Such air contains more oxygen and less carbon dioxide than
does alveolar air, but, on the other hand, it contains less oxygen and more
carbon dioxide than the expired air; it represents a mean between the two.
It is essential in such experiments that the gas tension of the arterial
blood should be ascertained at the same time and with the same individual.
Bohr experimented with large dogs which he compelled to breathe through
easily movable valves. A gas-meter measured the amount of the expired
air, from which a sample was taken for analysis. Bohr noted the depth
of each inspiration and determined, after the death of the animal, the
volume of the trachea and the bronchial tubes. From these values he
computed the composition of the air at the bifurcation. The partial pressure
of the oxygen and carbon dioxide in this gas was thereby known. Simul-
taneously, the gas-pressure from the blood of an artery was measured, in
order to establish normal relations as far as possible. Bohr prevented
coagulation of the blood by injecting peptone solution, or leech extract,
and carried the blood })ack through a vein into the general circulation,
so that the result of the experiment was not influenced by loss of
blood.

Carbon Dioxide Tension.

Difference between

Nature of Air breathed.

(a) Air at the

a and b.

Bifurcation.

(b) Arterial Blood.

16 6

10.1

- 6.5

Atmospheric Air.

14 3

16.7

+ 2.4

34.6

17.4

- 17.2

14.8

27.6

+ 12.8

40 0

29.7

- 10.9

4.9% CO2, 18.8% O2

28 . 5 1 0.9

- 27.6

3.2% COj, 20.0% 0,

The results of these experiments showed, on the one hand, that the
oxygen tension of the arterial blood flowing out of the lungs is frequently
more than that of the air at the bifurcation and, on the other hand, in

' Skand. Arch. Physiol. 2, 236 (1891).

OXYGEN.

431

several cases the tension of the carbon dioxide in the blood was less than
that of the air at the bifurcation. The following table gives an idea of
the results obtained : '

Weight of

O2 absorbed per Kil-
ogram in 1 minute.

Oxygen Tension.

Difference
between
a and 6.

Animal.

(a) Air at the

(6) Arterial

Bifurratioti.

Blood.

Kilograms.

14.1

9.8

127

144

+ 17

15.5

10.6

131

105

- 26

18.9

14 I

95

101

+ 6

41.5

14.7

110

122

+ 12

26.0

13.6

116

106

- 10

14.7

7 1

130

144

+ 14

These results indicate that neither the passage of oxygen from the alveo-
lar air to the blood, nor of the carbon dioxide from the blood to the alveolar
air, can be accounted for by diffusion alone. Some forces must be at
work which tend to make the oxygen more active towards its absorption
by the blood than can be accounted for by the partial pressures of the
oxygen gas, and at the same time these forces enable the blood to give up
its carbon dioxide even when the pressure of this gas is greater in the
alveoli than it is in the blood itself. Bohr compares the lung with a gland,
and conceives of its activity as that of a secretion. He assumes that
the lung-cells have the power of temporarily uniting with oxygen and with
carbonic acid. In fact, P. Ehrlich ^ has proved that the lungs possess an
extraordinarily strong reducing power. He injected alizarin-blue into
animals, this being a dyestuff which becomes colorless on reduction. The
lungs of an animal freshly-killed were then found to be colorless, the blue
color being apparent only after exposure to the air for some time. Now
the lung tissue, like all other tissue, has its own metabolism. It con-
sumes oxygen and evolves carbon dioxide. Its reduction power, according
to Ehrlich's results, however, is so pronounced that it seems perfectly
plausible to speak of an oxygen-secretion in the sense meant by Bohr.

Bohr and Henriques ' also made the discovery, which is of itself very
remarkable, that the lungs take an uncommonly large part in the general

' Cf. Bohr: Handbuch der Phj'siol. p. 146. Bohr measured directly the oxygen ten-
sion at the hmg surface and compared this with the oxygen tension of the arterial blood.
There wa.s in this case a more considerable excess of pressure in favor of the blood.
Cf. L. Fr^d^ricq: Zentr. Physiol. 7, 33 (1893); 8, .34 (1897). Haldane and J. Lorrain
Smith: J. Physiol. 20, 497 (1896); and 22, 231 (1899).

' Sauerstoffbediirfnis des Organismus. Berlin, 1885.

' Oversigt. kgl. Danske Videnskabs-Selskabs forhandl. No. 1, 1897, Arch, physiol.
9, 590 and 710 a897).

432 LECTURE XVIII.

metabolism. They estimate that about one-third of the total metabolism
takes place in this organ. We can understand this active metabolism, by
assuming that it is capable of performing a particularly intense kind of
work, and it is indeed possible that it is here that the work of secretion
comes into play.

It must be admitted that the views of Bohr have been vigorously chal-
lenged. His results have been attributed to an insufficient equilibrium
being established between the blood and the alveolar air. Bohr himself
would not admit this; and now that fifteen years have passed since his first
important results were published, without the appearance of any data
which conclusively disputes it, we are compelled to place his opinions in
the foreground in our discussion of the gas-exchange which takes place in
the lungs; although the older assumption that the laws of gas diffusion are
sufficient to explain this phenomenon is, on account of its greater sim-
plicity, very attractive. It is, of course, not impossible, but on the other
hand extremely probable, that diffusion does in part account for some of
this exchange of carbon dioxide between the blood and the alveolar air.
Other factors are probably active at the same time, so that there is a very
active penetration of oxygen into the blood and removal of carbonic acid
from the latter.

Bohr calls attention to the following observations in support of his
assumption. In the Amphibia it is well known that, besides the lungs,
the skin serves as an important organ of respiration. In the case of frogs,
simultaneous determinations of the gas-exchange of skin and lungs showed
that the taking up of oxygen by the skin is independent of the total extent
of the metabolism. It is almost constant, and amounts to 43 to 60 cubic
centimeters per kilogram of body weight in an hour. The carbon dioxide
elimination on the other hand showed variations of from 92 to 179 cubic
centimeters per kilogram in an hour. The gas-exchange taking place in
the lungs is different. Much more oxygen is absorbed through the lungs
than by the skin, and the variations are much greater (51 to 390 cubic
centimeters per kilogram in an hour) . The elimination of carbon dioxide
may, in winter when there is a considerable absorption of oxygen, sink
nearly to zero. Krogh,' who first mentioned these facts, found further
that a carbon dioxide tension of but a few per cent, in the atmosphere
surrounding the skin, caused a considerable increase in the amount of
oxygen taken up by the lungs alone, while at the same time the amount
taken up by the skin might be diminished. This action of the lungs does
not take place, however, if the cutaneous branch of the vagus nerve is cut.
The respiration by the skin is, on the other hand, apparently indifferent
to the nervous system. Evidently the gas-exchange by the skin takes place
by diffusion, while pulmonary respiration is more in the nature of a secretion.

' Skand. Arch. Physiol. 16, 378 (1904).

OXYGEN. 433

A very considerable secretion of gases takes place in the swimming-
bladder of fishes. Biot/ who examined the gases in fishes which live at
great depths, found sometimes as much as 80 per cent of oxygen. Whereas
the oxygen tension of water at a depth of, say, 1.500 meters, amounts to
only about one-fifth of an atmosphere, the partial pressure of this gas in
the swimming-bladder is equivalent to that of 90 atmospheres. Moreau ^
has shown, moreover, that the oxygen content of these gases in the swim-
ming-bladders of fish depends upon the depth at which the fish lives.
Those living near the top of the water often contain a lower oxygen pressure
than that of the atmosphere. If the same fish be placed at a greater depth,
it is no longer in equilibrium with its surroundings. Equilibrium is
restored, however, by more oxygen being secreted in the swimming-bladder.
If the bladder is emptied by means of a trocar, it refills with oxygen after
a time. This secretion, furthermore, is under the influence of the nervous
system. On severing the pneumogastric (vagus) nerve, the gas secretion
entirely ceases. Then, on artificially emptying the swimming-bladder,
it does not refill with oxygen. The epithelium of the bladder itself is
impermeable to oxygen. The oxygen passes out through the so-called
oval.

These observations are sufficient to prove beyond question that the
animal organism possesses cells whose function it is to secrete gases. It is
true that these results cannot be applied immediately to higher organisms,
but it gives undoubted support to Bohr's opinions.' By means of this
active taking up of oxygen, the animal organism obtains a certain supply
of this important gas, so that air containing but little oxygen suffices for
its support within certain limits. Thus, for example, muscular effort
requires an increased oxygen supply by an increase in the blood circula-
tion. In a unit of time more blood passes through the lungs. If, by an
artificial restriction of one of the pulmonary branches, more blood is made
to pass through one lung than through the other, there is more oxygen
taken up in the lung with the more blood, although the effect upon the
elimination of carbon dioxide is not so marked.^

An interesting question, but one not so easy to answer, is whether the
lungs of mammals are dependent upon certain nervous influences. This
is known to be true in the case of the tortoise. With the Tesludo grceca,
the trachea divides so high up in the neck that, without fear of injuring
the important nerves, cannulas may be placed in the bronchi, and thus
either lung be observed independently. If the vagus branches to one lung
are cut, the absorption of oxygen by that lung is lessened, while that of

M^moires de la soci^t^ d'Arcueil, 1807.

M^moires de Physiologie. Paris, 1877.

C. Bohr: J. Physiol. 15, 494 (1894).

V. Maar: Skand. Arch. Physiol. 15, 1 (1903); 16, 358 (1904).

434

LECTURE XVIII.

the other lung is increased.' The carbon dioxide elimination is similarly
affected.

Oxygen Absorption

n-

Right Lung.

Left Lvnig.

Total.

Kifect of severing the right vagus

Effect of severing the left vagus

j 15.4
1 30.0
I 29.1
1 21.4

17.1
5.3
5.2

14.9

32.5
35.3
34.3
36.1

Stimulation of the vagus leads to the opposite effect. It is not so easy
to decide how much these results are due to an influence upon the lungs,
and how much is due to the influence of the vasomotor fibres. Apparently
the latter is not sufficient to account for the whole phenomenon. The
influence of the vagus nerve has also been observed with mammals. Stim-
ulation of this nerve tends to make the I'espiratory quotient approach the
value 1.

Now if we consider once more the gas-exchange in the lungs, we see that
two processes are taking place side by side. Oxygen diffuses from the
alveolar air, which is relatively rich in this gas, and saturates the venous
blood with this element that is so important for the whole metabolism.
Simultaneously, the blood laden with carbon dioxide gives up the latter
to the alveolar air, which contains relatively less of it, and this takes place
until the partial pressure of the carbon dioxide in the alveoli is equal to
that of the blood, i.e., until equilibrium has been established. Now begins,
without doubt, the activity of the epithelium of the lungs by means of
which oxygen from the alveolar air is secreted in the blood-vessels, and has
the effect of overbalancing the equilibrium between the oxygen tension of
the blood and that of the alveolar air, in favor of the former. The carbon
dioxide is eliminated with equal avidity, and given up to the alveolar air.

Oxygen now circulates anew with the blood to the tissues whose oxygen
content is relatively less than that of the blood, so that oxygen is con-
stantly diffusing from the blood, and first of all the oxygen is lost, which
is merely dissolved in the blood. When this dissolved oxygen is lost, the
reserve supply, i.e., that combined with the oxyhemoglobin, comes into
play. The oxyhemoglobin now dissociates and oxygen is given up to the
plasma, in order to keep the oxygen tension of the blood up to a cer-
tain value. Now the question arises whether this internal respiration
takes place in accordance with the gas diffusion laws, or whether we must
assume that here also a secretion, i.e., an active giving up of oxygen,

• V. Maar: Skand. Arch. Physiol. 13, 269 (1902).

OXYGEN. 435

comes into play. At this point we meet with great difficulties in our
search for knowledge. To answer this question we must know, in the first
place, exactly how great the gas tension in the tissues is. We know, from
the experiments of Strassburg,' what the oxygen tension of the lymph is,
and as this surrounds all tissues and cells of the body, we get some idea as
to the gas-pressure prevailing there. Strassburg found the oxygen ten-
sion of the lymph greater than one atmosphere. According to the general
conception, the oxygen tension in blood is less than one atmosphere.
If this be true, it is necessary to assume some special activity as the
cause of the giving up of oxygen to the tissues. On the other hand, Strass-
burg found that the tension of carbon dio.xide in the lymph was less than
that of venous blood. From this fact we should speak of gas secretions
in the tissues. Some idea of the consumption of oxygen by the tissues
is obtained by tracing the oxygen tension of the blood in its transformation
from the arterial to the venous condition. -

The oxygen supply of the tissues may be regulated in quite a number of
different ways. The rate of the blood flow has an effect and again the
change of the content of the blood in hemoglobin, whether it be due to the
formation of new hemoglobin, or a relative increase by elimination of
plasma. By means of such changes combined with variations in the
intensity of work of the organs of respiration, the animal organism is,
within certain limits, independent of quite considerable variations in
oxygen and carbon dioxide tensions. It is highly interesting that each
lung has its own independent gas-exchange, and yet is able to mutually
compensate the other. If there is a greater oxygen tension in the air
supply of one lung, then a greater absorption of oxygen takes place in
this lung than in the other. At the same time, the other lung absorbs
less than the customary amount of oxygen, so that the total absorption
of oxygen by the two lungs remains about the same.

If the partial pressure of the oxygen in the inspired air becomes lower,'
then naturally that of the alveolar air becomes similarly affected. The
degree of change in the composition of the latter depends materially upon
the amount of oxygen absorption and the ventilation of the lungs. This
is an extremely important fact. It is for this reason that with the same
oxygen partial pressure, the alveolar air of two different individuals may
have a quite different composition, according as to whether one breathes
more deeply than the other, so that the lungs have a greater ventilation.

' Pfliiser's Arch. 6, 6,5 (1872).

' Cf. Ch. Bohr: Handbuch d. Physiol, p. 19G. Loewy and von Schrotter, Z. exper.
Path. u. Ther. 1, 197 (1905).

^ Cf. Paul Bert: La pression barom^trique. Paris, 1878. Frankel and Geppert:
Ueber die Wirkungen der verdiinnten Luft auf den Organismus, Beriin (188.3). A.
Loewy: Untersuchen iiber die Respiration und Zirkulation bei Aenderung des Druckes
■und des Sauerstoffgehaltes der Luft, Berlin, 1895.

436 LECTURE XVIII.

The lower limit for oxygen tension in the alveolar air lies a little above
30 millimeters. This corresponds to an oxygen content of about 4 . 5 per
cent, assuming a total pressure of 710 millimetres (= 1 atmosphere at the
body temperature) . Moreover, this is true only for a period of rest, and
not for one of active work. In the latter case an oxygen pressure of
30 miUimeters is not sufficient.

Paul Bert, Frankel, and Geppert have shown that the amount of oxygen
absorbed by the blood becomes equal to one-half the normal amount,
only when the total pressure of the surrounding atmospheric air is less than
300 miUimeters. This is interesting, because it gives us some conception
as to the nature of the behavior of the gas-exchange during passage into a
more rarefied atmosphere, i.e., in balloon ascensions, or in mountain climb-
ing. In these two examples naturally the requirements upon the blood-
gases are quite different. In the former case, there is practically no work
to be performed, so that aeronauts reach a much higher altitude than do
mountain climbers, before they experience difficulty in breathing. The
fact that different individuals are affected differently at one and the same
height is explained, first, by the fact that the lung ventilation and amount
of air breathed is, as already mentioned, quite different, so that, in one
case, the blood has more oxygen at its disposal than in another. It has
been found, moreover, that the animal organism possesses an extremely
delicate mechanism of regulation, which energetically opposes any defi-
ciency of oxygen in the system. To this belongs the increase in the number
of red corpuscles, and thereby of hemoglobin, which unquestionably takes
place when men and animals pass from a locality into one of higher altitude.
The increase disappears as soon as the original level is again reached.'
The object of this is plain. No matter whether we assume that the abso-
lute amount of these red corpuscles is increased, or that the increase is
merely relative, brought about perhaps by the passing out of plasma,
there is in a unit of blood more hemoglobin passing through the lungs in
a unit of time than is normally the case. The way this increase in the red
corpuscles caused by ascending high mountains is effected, has not been
satisfactorily explained. It is remarkable that the change takes place
suddenly, and in fact without any indication of there being any new forma-
tion of blood (red corpuscles with nuclei, etc.), and that on reaching a low
level again, the reverse change takes place without any of the usual indi-

' Cf. Paul Bert: he. cil. Die histiochemischen und pliysiologischen Arbeiten von
Fr. Miescher, Vol. II, p. .328, Leipzig, 1897. Abderhalden: Z. Biol. 43, 125 and 443
(1902); Medizin, Klinik, No. 6 (1905); Pfluger's Arch. 110, 195 (1905). von Schrotter
andZuntz:*i(i. 92, 479 (1902). van Voornveld: i6i(i. 92, 1 (1902). Otto Cohnheim:
Ergeb. Physiol, (.\sher and Spire) II, 612 (1902). Durig and Zuntz: Arch. Anat.
Physiol. Suppl. 1904, p. 417. Jaquet: Ueber die physiologische Wirkung de.s Hohenkli-
mas, Basel, 1904. Zuntz, Loewy, Miiller, and Caspari : Hohenklima und Bergwanderungen
in ihrer Wirkung auf den Menschen, Bong et Cie, 1906.

OXYGEN.

437

cations of a diminution in the number of red corpuscles. There is abso-
lutely no doubt that after staying at a mountain height for some time, an
acclimatization takes place in the sense that a new formation of hemo-
globin results. It remains undecided how much this is due to an abso-
lute increase in the number of red corpuscles, and how much to a merely
relative increase. It seems reasonable to believe that this may be brought
about by the adjustment of the vascular tonicity to definite pressures
of the atmosphere.'

It remains for us to decide whether, besides the lungs, other organs of
the body take part in the gas-exchange. We have already seen that with
Amphibia respiration on the part of the skin plays quite an important part.
In higher vertebrates this cutaneous respiration does not seem to be
hardly worth considering. Schierbeck - estimated that in man there is
an elimination of carbon dioxide amounting to 9 grams per 24 hours,
or somewhat less than 1 per cent of the total gas-exchange. If there
is an increased secretion of sweat, it may rise as high as 30 grams in
24 hours. The absorption of oxygen is much less. The following table
prepared by Krogh ^ gives a good idea as to the extent of this cutaneous
respiration on the part of man and certain animals:

O2

COj

Maximum.

Average.

Maximum.

Average.

Man

Man

0.92

0.1

1.8

2.1

1.05

0.50

0.47

1.51
1.62
0.74

3.V

1.1

0.15

5.3

4.4

1 18
0.94
0 60

Tortoi.se

3 0

Rana esculenta

Eel

3.1

The above values refer to the amount per hour and per square decimeter
of the skin. The volumes of the gas are given in cubic centimeters.

It was for a long time believed that the skin took part in the elimination
of the gaseous products of metabolism. It had been observed, for example,
that if the skin of an animal were varnished over it soon died. This,
however, has more recently been found to be caused not so much by the
retention of waste gases, as by the greatly increased amount of heat,
caused by the crippling of the means for regulating the body tempera-

' Mountain sickness has been attributed to various causes, one of which is undoubt-
edly a faulty regulation of the vascular tone.

= Arch. Anat. Physiol. 1893, 116. Cf. Aubert: Pfluger's Arch. 6, 539 (1872).
' Skand. Arch. Physiol. 16, 378 (1904).

438 LECTURE XVIII.

ture. If this increase in temperature be prevented, the animal will not
die.'

We recognize certain kinds of fish, especially the loach, Cobitis fossilis,
in which there is a peculiar intestinal respiration. The middle intestine
of the Cobitis contains an abundant supply of capillary blood-vessels and a
peculiarly transformed epithelium. These fish swallow air, and discharge
gases through the rectum. The gas which leaves the body contains less
oxygen and more carbon dioxide than that entering.^

With the remaining members of the animal kingdom, the intestine plays
no part in the gas-exchange. To be sure, the alimentary canal contains
gas, resulting, in part, from swallowed air, which with the food, the saliva,
and drink, is constantly being introduced, and largely from bacterial
decomposition, fermentation, etc. Furthermore, carbonic acid is set free
in the neutralization of the carbonates of sodium in the intestinal secretions
by the hydrochloric acid from the stomach. The oxygen of the swallowed
air is taken up little by little by the intestinal walls; and similarly, on
account of its partial pressure, the carbon dioxide diffuses to some extent
into the intestinal walls and into the blood-vessels, and in other cases
these gases are given up from the vascular system, if the amount present
in the intestine is slight. Likewise other gases, such as hydrogen, methane,
sulphureted hydrogen, and nitrogen, are absorbed according to the laws
of gas absorption, and can be ehminated by the lungs. ■

' Cf. Laschkewitsch : Arch. Anat. Physiol. 1868, 61. R. Wintemitz: Arch, exper.
Path. Pharm. 33, 286 (1895). E. BabAk: Pfliiger's Arch. 108, 389 (1905).

' Bauraert: Chemische Untersuchungen der Respiration des Schlammenpeizgers,
Breslau, 1855. D. Calugareanu: Pfluger's Arch. 118, 42; 120, 425 (1907).

LECTURE XIX.
ANIMAL OXIDATIONS.

In the last lecture we attempted to trace the path of oxygen on its way
through the animal organism from the time of its being acquired from the
alveolar air to its being given up to the tissues and their cells, and, on the
other hand, we found that carbon dioxide is to be regarded as the end-
product in the action of oxygen upon the nutriment. Meanwhile, we have
failed to touch upon one point of greatest moment, namely, why the oxygen
attacks and consumes this cell-nutriment. Outside the animal organism,
if we expose albumin, fats, or carbohydrates to the action of oxygen at
the body temperature, even for a long time, there is no perceptible oxida-
tion of these materials. Within the animal organism, on the contrary,
these substances are oxidized in a short time, and the chief products of the
oxidation are carbon dioxide, water, and urea. Consequently, conditions
must prevail within the organism which facilitate the action of oxygen
upon the material exposed to its action.

We are acquainted with quite a number of facts which compel us to
assume that, even within the animal tissues, oxygen as such is not able to
act upon the unchanged food. Under no circumstances should we imagine
for a moment, that the oxygen supplied to the tissues, at once of its own
accord, begins to oxidize the different substances present in the cells. If
this were the case, it would be absolutely impossible for us to account for
quite a number of processes taking place in the animal organism. Above
all, it would then be unintelligible, why oxygen is brought to the cells,
together with the newly absorbed nourishment, without any oxidation
taking place until the cells are reached. The fact that the blood contains
the greater part of its oxygen chemically united with the hemoglobin, does
not suffice to explain this fact; for it would be expected that the oxygen,
which was merely dissolved in the plasma, would be replaced, as soon as
consumed, by the oxygen in the hemoglobin. Again, it would be inex-
plicable why, in the combustions taking place in the cells, it is only the fuel
that is consumed, and not the cell-substance itself. On the other hand, we
have seen that the organism can lose the power of oxidizing certain sub-
stances, such as carbohydrates, for example, which are ordinarily consumed
easily, while other oxidation processes are not affected in the slightest.
We know now that in diabetes substances hard to oxidize are consumed
without difficulty; whereas, unchanged d-glucose alone has ceased to be a

439

440 LECTURE XIX.

food, because the organism has lost the power of being able to utilize the
energy stored up in it. If, on the other hand, the grape-sugar is slightly
changed before its introduction into the organism of the diabetic, then the
tissues are capable of completely oxidizing it.

If the animal oxidation took place merely as a result of the coming
together of oxygen and nutriment, it would be expected that when an
increased amount of the reacting substances was present, a more vigorous
oxidation would ensue. This is, however, not the case. Under normal
conditions, it is not possible to increase the amount of oxidation taking
place in the tissues by increasing the supply of oxygen and the amount
taken up by the blood.' Similarly, we are not able to increase the total
consumption of material very much by increasing the supply of carbo-
hydrate or fat. Only of albumin do we know that the amount present
governs somewhat the extent of the transformation.

We know, to be sure, of compounds which are not attacked by oxygen
in neutral solutions, but are attacked in the presence of alkali. Pyrogallol
absorbs oxygen in alkaline solutions so vigorously that it is used for the
detection of small quantities of this gas. To be sure, we do not have free
alkali present in our tissues, but merely alkali carbonates. These also favor
such oxidations. Thus it is known that a solution of glucose and soda
absorbs oxygen from the air,^ although the amount taken up is but slight.
Schmiedeberg ' has shown, moreover, that benzyl alcohol in the presence
of water is not attacked by atmospheric oxygen, but is transformed to
benzoic acid when it is in a sodium carbonate solution exposed to the oxy-
gen of the air. This compound is also oxidized by the oxygen in the blood.
If benzyl alcohol is conducted, with blood containing oxygen, through the
kidneys or lungs of dogs or pigs, benzoic acid is formed; while if salicylic
aldehyde is employed in the above experiment, salicylic acid is formed to
some extent. The amount of acid obtained in each case is very small. It
is necessary to state in this connection that these experiments are by no
means sufficient to account for the combustion of the nutriment. They
merely show us that easily oxidizable substances are more readily acted
upon by oxygen, when contained in alkaline solutions, than in neutral or
even acid ones. They in no way refer to the oxidation of the more diffi-
cultly oxidizable foodstuffs.

We are compelled to assume that either the oxygen is changed in the
tissues to a form in which it is more active than usual, or that the nutriment
is in some way changed by the activity of the cell, so that it is more readily
acted upon by oxygen. Or these two processes may take place side by
side in the cell.

' See Schatemikoff: Arch. Anat. Physiol. 1904, Suppl. 135.
' M. Nencki and N. Sieber: J. pr. Chem. 26, 1 (1882).
' Arch, exper. Path. Pharm. 14, 288 (1881).

ANIMAL OXIDATIONS. 441

The fact that the oxygen in the condition in which it is given up to the
tissues is not capable of consuming the unchanged nutriment, enables the
cell to adjust the metabolism to its requirements. Above all, this fact
enables the cell to utilize a certain particular material for definite functions
when it so desires. We should also not forget that we know altogether too
little concerning the relations of the separate organs to one another, and
of the different kinds of cells in one and the same organ, to be able to
judge whether under all conditions, the combustion of the fuel is entirely
efTected by the cell that begins the work, or whether one cell merely
carries the oxidation to a certain stage, and another cell carries the com-
bustion farther, until finally the material is completely oxidized. Such
an assumption seems extremely probable from the observations of Bohr
and Henriques,' who found that extensive oxidations take place in the
lungs. Oxygen is consumed there and carbon dioxide evolved to an extent
sufficient to lead one to presume that incompletely oxidized, metabolic
products reach the lungs together with the blood, and that the combustion
is completed by the lungs. On the other hand, according to the assump-
tion that the respiratory exchange takes place as a sort of secretion process,
it is probable that the lungs have a certain amount of work to do which
requires the expenditure of energy, so that this supply of fuel is necessary
for its function. Bohr and Henriques found that about one-third of the
total metabolism taking place in the body was effected in the lungs. The
separate observations varied from 0 to 66 per cent.^

We must emphasize the fact at the start that we are not yet able to give
a perfectly clear explanation as to the nature of animal oxidation processes.
At present, we recognize merely the initial products, food and oxygen, and
the final products of the comlmstion. We are therefore forced to rely
upon assumptions. In fact, the great number of hypotheses which have
been brought forward, show clearly upon what an insecure foimdation they
all rest. Here we will only attempt to mention only the more important
theories, and emphasize only those which i-est, to some extent, upon
experimental observations.

The question to interest us first of all is this: Is the oxygen changed
in form so that it attacks the nutriment more readily? The formation
of ozone has been suggested. We know that ozone is a stronger oxidizing
agent than ordinary oxygen, and oxidizes compounds which are not
affected by the latter. Schonbein' carried his studies on ozone to the
phenomena of cell life. He attributed numerous oxidations taking
place in the plant organism to the primary formation of ozone. The plant
tissues, he assumed, contain some substance which possesses the power of

' Arch, de Physiol. 1897, 590. See Lecture XVIII, p. 432.

' These high figures have not been generally accepted. — Translator.

' Poggendorff's Annalen, 65, 171 (1845).

442 LECTURE XIX.

ozonizing atmospheric oxygen. The cells take up the ozone, and then as
the latter breaks down to ordinary oxygen, the extra atom of oxygen
attacks some oxidizable substance. The assumption that as a matter
of fact there is an ozone formation in the tissues, meets with certain diffi-
culties. Relatively small amounts of ozone are poisonous to the cells. It
has never been possible to detect the presence of ozone in either plant or
animal organisms. So this hypothesis was soon abandoned. The as.sump-
tion that active oxygen is present in the tissues, is far more probable.
Hoppe-Seyler,' who suggested this hypothesis, based it upon the fact,
that in the animal tissues energetic reduction processes take place, side by
side with the oxidations. In this way, reducing substances are formed
which unite with one atom in the oxygen molecule, setting the other atom
free in an active condition. The butyric acid fermentation of sugar is an
example of such a reduction process. Hydrogen is set free:

CeHioOo = C3H7COOH + 2 CO2 + 2 H2.

A support for the assumption that active oxygen causes the animal
oxidations, is furnished by the theory of nitrification. In this case also,
it is assumed that the organism, to whose activity the formation of nitrate
is due, first of all produces readily oxidizable substances, which then decom-
pose the atmospheric oxygen molecule, and thus form active (nascent)
oxygen for the oxidation of the nitrogen.

The formation of such readily oxidizable substances which under normal
conditions are immediately oxidized further, is likewise indicated by the
so-called " spontaneous combustion " of hay. In this ease, on account
of insufficient ventilation, such readily oxidizable substances collect in
considerable amount, and are suddenly oxidized as soon as fresh air
enters.

If we are to believe that the oxidations in the animal organism take
place in this way, we must assume that first of all the food is hydrolyzed,
and that easily oxidizable compounds are formed which are oxidized by
the oxygen, received by the tissues from the blood, and at the same time
a part of this oxygen is activated. This nascent oxygen unites with the
more difficultly oxidizable substances. The first stage in the oxidation
of the food is, therefore, a hydrolysis; and inasmuch as the last-mentioned
process is brought about by means of ferments, these play a part in the
entire phenomenon of oxidation. This assumption has much in its favor,
and corresponds to certain discoveries. With its help we are able to under-
stand, for example, why the diabetic cannot oxidize rf-glucose, and only
this one substance. We have simply to assume that the ferment is absent
which hydrolyzes this sugar so that oxygen does not come in contact with

» Pfluger's Arch. 12, 10 (1S70).

ANIMAL OXIDATIONS. 443

the cleavage-products of this substance. To be sure, this does not explain
why the nascent oxygen set free in the combustion of other foodstuffs,
is not able to act upon rf-glucose. At all events, the assumption that oxi-
dation is preceded by a hydrolysis, gives to the cell the power of utilizing
its nutriment at the time it is needed. We know that the action of the
ferments is specific, i.e., that they are able to act only upon certain definite
compounds. If it be assumed that the cells do not contain the ferment
in an active condition, but that the ferment is activated only when its
action is needed, we are able to understand very clearly much about the
economy of the cells. We begin to understand how the cell can have
the food, the ferment, and oxygen all present together without any com-
bustion taking place, until at a given moment the ferment becomes
activated. The following phenomenon harmonizes well with such an
assumption.

The animal organism consumes without difficulty the cleavage-products
of the food which it obtains as such. Thus the decomposition products of
albumin, such as glycocoll, alanine, etc., are readily oxidized to urea.' This
is true, however, only of those amino acids which are present in albumin,
i.e., those having the same configuration. If, for example, instead of feed-
ing a rabbit with ^-leucine, we administer the racemic form, rf-Weucine,
only a part of the molecule, namely the /-leucine, is oxidized, while the
other half of the racemic substance molecule, the right-rotating leucine,
appears unchanged in the urine. This is evidently because the animal
cells are not adapted to the combustion of d-leucine, which under ordinary
conditions is foreign to it, so that it possesses no ferment to attack it.
The oxygen, therefore, is not able to attack it. Furthermore, it is not
strange that when the combustion is very vigorous a part of this substance
is actually oxidized. We may indeed assunie that oxygen activated by
some other decomposition process can cause such oxidation.

Interesting as this hypothesis appears, we must not forget to state that
in this simple form it does not suffice to explain all the phenomena pertain-
ing to animal oxidation. The actual relations are far more complicated
than we have indicated. Above all, we would expect that in the absence
of oxygen there would tend to be a piling-up of these readily oxidizable sub-
stances, and to a considerable degi-ee, particularly as now the entire energy
required by the organism must l^e furnished by the hydi'olytic decomposi-
tions. We have already mentioned experiments performed in this direc-
tion.^ G. von Bunge^ has shown that ascarids are able to exist for
several days without any oxygen supply. During this time they move
about quite actively. The energy necessary in such cases must be pro-

■ Cf. Lecture XI, p. 228.

^ Lecture IV, p. 74.

3 Z. physioL Chem. 14, 318 (1890).

444 LECTURE XIX.

duced by means of partial decompositions. We should expect, therefore,
that hydrogen and other easily oxidizable substances would be formed.

This was, however, not the case. The presence of hydrogen could not be
detected, nor did oxygen disappear, if oxygen was supplied after the worms
had existed for a day without it. Now we have no right to apply the
results of such experiments to human beings, or to other animal organ-
isms. The parasites of the intestine are accustomed to get along with a
very limited supply of oxygen. It is perfectly possible that their metabo-
lism takes place entirely differently than is the case with the remaining
animal organisms. Perhaps hydrolytic decompositions take place in
them without forming any easily oxidizable substances. Furthermore,
it is conceivable that these little worms normally have active oxygen at
their disposal, formed, at least to some extent, by the energetic reduction
processes which take place in the alimentary canal. On the other hand,
•we must remember that the assumption of an activation of oxygen, by
means of the formation of reducing substances in the tissues, has never
gotten beyond the hypothetical stage. It is i-emarkable too that the
animal organism itself is able to keep easily oxidizable substances in its
tissues, such as phosphorus, for example, in an unchanged condition for
a considerable length of time. It is not easy to understand how such a
substance as this escapes the active oxygen unless it be assumed that
during its transport through the animal tissues, it never actually
comes in contact with nascent oxygen. Such an assumption seems
strained, for we know of countless examples in which the animal organism
is protected from poisons by their being oxidized. The oxidation does not
always take place to such an extent, but frequently it serves to prepare a
new substance which can become harmless by conjugation with something
else, whether it be glycocoll, sulphuric acid, glucuronic acid, or urea. On
the other hand, it is a fact that a reduction process may facilitate the
combination of the poison with one of the above compounds. At all
events, it is extremely interesting to find that these oxidations are quite
different in various cases. At one time the compound may be completely
oxidized, whereas, in another case, merely an atom of oxygen may be
added to its composition. We can, indeed, believe that the cells, as
already mentioned, may regulate their decompositions by means of their
ferments; but it remains an enigma, according to the assumption of the
presence of active oxygen, why the oxidation should stop at a certain
stage in the case of readily oxidizable substances, whereas in other cases
a total oxidation takes place.

Unquestionably, we would prefer an hypothesis which in itself includes
this regulation of the extent of oxidation. The first suggestion of such an
hypothesis was made by Moritz Traube.' He pointed out the importance

' Theorie der Fermentwirkungen. Berlin, 1858.

ANIMAL OXIDATIONS. 445

of oxygen carriers. In the study of inorganic chemistry, we know of
various processes which take place in the presence of a carrier of oxygen,
but will not take place from direct contact with oxygen. An e.xample of
such a process is in the oxidation of glucose by potassium indigo sul-
phonate, or by copper oxide. If a solution of glucose is warmed with
alkali carbonate in the air for some time, practically no oxygen is taken
up. As far as we can see, the glucose remains unaffected. Now if we add
a little potassium indigo sulphonate to the solution, in the course of a
short time the blue solution becomes decolorized, and the amount of
unchanged glucose remaining in the solution gradually becomes smaller
and smaller. The glucose has become oxidized. If now we shake the
solution, it soon turns blue again, but again on standing it becomes color-
less. At the same time, more of the glucose becomes oxidized. This
process may be repeated again and again, until finally no more glucose
remains. A very small amount of potassium indigo sulphonate suffices
to cause the oxidation of a large amount of glucose. Copper oxide exerts
a perfectly similar effect. An ammoniacal solution of cupric oxide
becomes decolorized on warming it with glucose. The cupric oxide is
converted into cuprous oxide, or, in other words, the copper is reduced.
Oxygen has been furnished to the glucose molecule. If the decolorized
solution is exposed to the air for some time, gradually, and first at the
places where the solution is in direct contact with the air, it turns blue
again. Oxygen is thus being taken up by the cuprous oxide, which may
be given up subsequently to more glucose. The whole process can be
accelerated considerably by shaking the liquid with air.

It is not difficult to conceive that substances are present in the animal
tissues which act as carriers of oxygen for the more difficultly oxidizable
substances. As we have said, a very small amount of such substances
suffices to accomplish an indefinite amount of oxidation. The carrier
itself is in the same condition at the end of the process that it was at the
beginning. Now we know that ferments play an important part both in
the animal and vegetable kingdoms. The thought naturally arises that
perhaps certain of these ferments may act as carriers of oxygen. Traube
speaks of oxidizing ferments. Recently Schmiedeberg ' has mentioned
the possibility of certain definite oxygen-carrying ferments; while Jaquet ^
has followed up this suggestion and succeeded in proving that extracts
of the organs serve as carriei's of oxygen, and that in fact the principle
which causes this action may be precipitated by means of alcohol without
its losing any of its power of causing oxidation. Heating to 100° C,
however, destroys it.

To-day, no one doubts that a great many such ferments are actually

' Arch, exper. Path. Pharm. 14, 288 (1881).
' Ibid. 29, 386 (1892).

446 LECTURE XIX.

present, both in the animal and vegetable kingdoms. It is easy to detect
their presence by means of certain chemical reagents. If, for example,
an organ extract is shaken with an alkaline solution of a-naphthol plus
p-phenylenediamine, the formation of blue indophcnol is soon apparent.'
Without the addition of the organ extract, the formation of this color
takes place very much more slowly. A reaction made use of by Schonbein
is the blue color obtained with tincture of guaiacum. Its indication of the
presence of oxidizing ferments is, however, not altogether reliable. The
guaiacum reaction is also brought about l>y the presence of numerous other
oxidizing agents, such as ferric chloride, chromic acid, chlorine, bromine,
etc. By means of these organ extracts salicylic acid may be converted
into benzoic acid, and benzyl alcohol into benzoic acid.^ Formaldehyde
is similarly converted into formic acid,^ and arsenious acid into arsenic
acid.* a-naphtylamine is changed into violet-blue oxynaphtylamine, and
benzidine into a brownish-violet substance.' Likewise phenolphtalin is
changed to phenolphthalein." These are a few examples of the oxida-
tions which may be brought about readily by means of organ-decoction or
organ-extract; and, in fact, with plants the oxidizing action of certain of
their organs, the roots for example, may be demonstrated directly by
allowing them to grow upon strips of paper moistened with solutions of
the above-mentioned reagents.

By means of these discoveries, the whole question of animal and vege-
table oxidations, the latter not differing from the former in its essential
particulars, has been turned in an entirely new direction. Although,
at the present stage of its development, it is perhaps going too far to speak
of a perfect explanation of the oxidation processes in the tissues, still, on
the other hand, it is true that numerous processes which would be other-
wise beyond our comprehension, are now better understood. As we shall
see later, we have every reason to believe that a given ferment always acts
upon only quite definite compounds. Thus the proteolytic ferment, tryp-
sin, attacks only proteins, and not carbohydrates. It might have been
assumed a priori, that the oxidizing ferments are exceptions in this respect,
and are in general capable of serving as carriers of oxygen. This is, as a
matter of fact, not the case, as we know from numerous observations.
Thus it is only certain definite oxidizing ferments which are capable of
causing the above-mentioned oxidation of a-naphthol and p-phenylene-
diamine to indophenol in alkaline solution. In the liver, for example,

' F. Rohmann und W. Spitzer: Ber. 28, 567 (1895).

' Jaqiiet; Arch, exper. Path. Pharm. 29, .386 (1892).

' Pohl: ibul. 38, 65 (1896).

' W. Spitzer: Pfliiger'.s Arch. 71, 596 (1898).

' M. Raciborski: Anzeiger der akad. der Wissensch. zu Kraukau, 1905, 338.

' Kastle and Shedd: Am. Chem. J. 26, 527 (1901).

ANIMAL OXIDATIONS. 447

the ferments belonging to this organ are capable of converting aldehydes
(e.g., salicylic aldehyde) into the corresponding acid, but they are inca-
pable of effecting the indophenol synthesis. Jacoby ' was unable to effect
the oxidation of such compounds as acetic acid, stearic acid, etc., by
means of the ferment which changes salicylic aldehyde into salicylic acid.
Again, ferments are known which will turn tincture of guaiacum blue, but
have no action upon salicylic aldehyde. -

Particularly in the vegetable kingdom • we meet with these ferments
possessing a quite specific oxidation power, which are designated with
particular names according to the work that they perform. In the
sap of plants we often find the so-called tyrosinase^ i.e., a ferment
which transforms tyrosine into colored products. The same, or at
least a very similar ferment, also occurs in animal organisms. Thus
the stomach juices of starved meal-worms act upon tyrosine.^ A
similar action explains an old observation. As is well known, the blood
of insects is nearly colorless, but becomes dark as soon as it leaves the
body. Von Fiirth and Schneider ^ have found that this so-called melanosis
is the result of the action of an oxidizing ferment. They proved this in
the following manner: They obtained, by piercing the pupae of Deilephilia
elepenor and euphorbia:, a greenish-colored liquid, from which they obtained
a precipitate upon the addition of ammonium sulphate. This precipitate,
on being dissolved in 0.05 per cent soda solution, and added to a solution
of tyrosine, soon caused a violet coloration, which gradually turned black.
Eventually dark flocks of a precipitate were thrown down. The tyro-
sinase thus obtained acts upon other aromatic compounds, containing
hydroxyl groups, such as catechol, chinol, etc. In the insect blood itself,
tyrosine is not present, but a chromogen, which is evidently closely related
to it. It may be assumed safely, that tyrosinase is very abundant in
nature, and undoubtedly plays an important part in the formation of pig-
ments. In the blood of the river-crab and in the ink-glands of the cepha-
lopoda a tyrosinase is found.

Tyrosinase is much more widely distributed in the vegetable kingdom
in which it was discovered, Bertrand " has found besides the tyrosinase
a second ferment, laccase, which acts upon only quinol and pyrogallol.

Closely related to these ferments is the glucolytic ferment, which we
have already discussed,' but whose existence, however, has not been posi-

• Virchow's Arch. 157, 235 (1899).

' Abelous et Biarnes: Compt. rend. soc. biol. 49, 175, 285, 493, 559, 596; 50, 495.

' E Bourquelot and G. Bertrand: J. pharm. chim. (6) 3, 177 (1896); Bull. soc. mycol.
France, 1896, 18 and 27.

« W. Biedermann: Pfluger's Arch. 72, 105 (1898).

« Hofmeister's Beitrage: 1, 229 (1901).

« Compt. rend. 122, 1132, 1215 (1895); 123, 463 (1896).

' See Lecture IV, p. 73, and V, p. 87.

448 LECTURE XIX.

tively established. The oxydase which takes part in breaking down the
nucleins also belongs here.'

In this class of oxidizing ferments we must also reckon the ferment of
the acetic acid bacteria, discovered by E. Buchner and J. Meisenheimer.^
Quite independently of the other activity of these microbes, this ferment
changes alcohol into acetic acid:

CH3 . CH2OH + 02 = CH3 . COOH + H2O.

Unquestionably, a great many other oxidations are to be traced to the
action of oxidizing ferments, and we find in the literature numerous other
ferments described under particular names. We must not forget, how-
ever, that the identification of ferments is an extremely difficult task, and
it is very seldom that in any case it is absolutely proven a new one is at
hand. Thus we know from the investigations of Bertrand ' that in the
juice of the berries of mountain ash a hexatomic alcohol, sorbitol, is present
which under certain conditions is changed to the hexose, sorbose. It would
be unjustifiable from this fact alone to assume that this evident oxidation
is to be attributed to the presence of a ferment. It has been, in fact,
established that a certain species of bacteria, Bacterium xylinurn, which
is introduced into the berries by means of a tiny red flea, Drosophila
funebris, causes this transformation. This bacterium will likewise oxidize
mannitol to fructose, xylose to xylonic acid, etc. Although it is indeed
very likely that these bacteria act side by side with oxidizing ferments,
still, on the other hand, it is not right to assume their presence until the
ferment itself has been isolated.

We have intentionally made this digression in order that we may gain
some idea as to the way in which such oxidations are effected, and as to
the abundance of such ferments. Of course this has not told us much
concerning the " oxidative " breaking down of the most important food-
stuffs. At present we do not know exactly how and when the oxidation
takes place in the case of protein, fat, or carbohydrate. We can make
assumptions, but we know nothing with certainty.

We have up to now concerned ourselves merely with the empirical
knowledge, and have not said anything as regards the way in which these
ferments act. Perhaps a glance at the mechanism of their action may
give us some idea of the oxidation as it takes place in the tissues. Unfor-

' See Lecture XIII, p. 293.

' Bar. 36, 6.34 (1903).

' Compt. rend. 122, 900 (1896); 126, 842, 984 (1898); 127, 124, 758 (1898). Con-
cerning the oxidation of glucose to gluconic acid, and of gluconic acid to oxygluconic
acid, see Bontroux: Ann. Pasteur, 2, 308 (1887); Compt. rend. 127, 1224 (1898). Oxi-
dation of quinic acid to protocatechuic. Emmerling and Abderhalden: Zentr. Bac-
teriol. Parasitenkunde und Infectionskrankheiten, 10, .\bt. II, p. 337 (1903).

ANIMAL OXIDATIONS. 449

tunately, we must again state that we are now dealing entirely with
hypotheses, and that we are not yet in position to explain with certainty
the exact nature of the processes which are brought about by the action
of oxidizing ferments.

Traube ' suspected the formation of hydrogen peroxide. In the oxida-
tion of readily oxidizable substances, instead of the oxygen molecule being
merely split and active oxygen set free, he imagined that water is first split
off. The hydroxyl group in the latter then combines with the oxidizable
substance, R, and the free hydrogen atom combines with neutral oxygen,
forming hydrogen peroxide:

R + 2 H2O + 0^ = R(0H)2 + H2O2.

Hydrogen peroxide is a strong o.xidizing agent, and will attack the diffi-
cultly oxidizable substances.

The above process may be supposed to take place somewhat differently.
Perhaps the readily oxidizable substance (R) combines with the oxygen,
and forms itself a peroxide. The latter can then give up its extra atom
of oxygen to some difficultly oxidizable substance (Ri).'

R + O2 = RO2. RO2 + Ri = RO -h RiO.

In this case the peroxide takes the place of the hydrogen peroxide, and
has the same effect. According to this theory it is not that we have active
oxygen present, but rather that the oxygen in the peroxide is held in a
loosely combined condition from which it is easily set free.

The question now arises. What connection do the ferments have with this
peroxide formation? We can imagine that the ferment itself combines
with oxygen, and by forming a peroxide thus acts as a carrier. In
fact, such an action has been assumed, and A. Bach with R. Chodat^ has
supported the a.ssumption by quite a number of experimental observa-
tions. These authors carried out their investigations with plants. They
separated the ferments which take part in oxidations into three classes.
First, there are albumin-like substances which form peroxides from the
oxygen that is brought to the tissues by the blood. Such ferments they
designate as oxygenases. Then Bach and Chodat identified peroxydases,
ferments which have the power of increasing the peroxidation power of
the former. Finally, in each cell there are present, according to these
authors, catalases, which decompose hydrogen peroxide catalytically with
evolution of oxygen.

' Ber, 10, 1111 (1886); 10, 1115; 22, 1496 and 3057 (1889); 26, 1471 and 1476
(1893).

' C. Engler and W. Wild; Ber. 30, 1669 (1897).

' Biochem. Zentr. 1, 417 and 457 (1903). X. Bach: Berichte, 38, 1878 (1905); 37,
3785 (1904). Bourquelot: Compt. rend, de la soc. biol. 49, 402 (1897). Batelli and
Stem: Corapt. rend, 140, 1197 and 1352 (1905); 141, 1.39 (1905).

450 LECTURE XIX.

Substances which act catalytically upon hydrogen peroxide have been
known in nature for a long time. It was even known to Thenard ' that
fibrin and the tissue of the kidneys and lungs were capable of decom-
posing hydrogen peroxide into water and oxygen just as energetically
as is done by platinum, gold, and silver. In milk^ also, and in blood,'
there is a hydrogen-peroxide-catalase. The catalases are evidently of
quite common occurrence.* Their significance is differently explained.
It is held that they tend to prevent the appearance of active oxygen. It
may be shown, for example, that urea and xanthine cannot be oxidized
by hydrogen peroxide in the presence of a catalase. They decompose
hydrogen peroxide with the formation of molecular oxygen.^ In fact, the
catalases are not oxidizing ferments at all. They are not of themselves
able to turn tincture of guaiacum blue, nor in the presence of hydrogen
peroxide. Thus the cells evidently possess a means of restricting and
regulating the activity of the oxidation processes taking place within
them.

The action of the oxygenases appeared to have a very simple explana-
tion, when it was found that their ash contained substances which of
themselves could act as carriers of oxygen. Thus Bertrand " found 2.5
per cent of manganese in the ash from laccase. Manganese salts are
very active carriers of oxygen. In other oxidases iron is present. Ber-
trand compared 0.1 gram of ferment in 50 cubic centimeters of quinol
solution in its action first with manganese alone and then with a man-
ganous salt plus the ferment.

Manganous salt alone 0.3 c.c. oxygen absorbed

Laccase, from Lucerne, alone .... 0.2 c.c. oxygen absorbed
Laccase plus manganous salt .... 6.3 c.c. oxygen absorbed

These inorganic constituents have been assumed to combine with albu-
min, and form a dissociable compound. The metal constituent serves to
carry the oxygen and in much the same way as we described for the
oxidation of sugar in alkaline solution by means of copper oxide. The
manganese is, according to this view, originally present in the divalent or
manganous form, which takes up oxygen from the tissues with the forma-
tion of tetravalent mangane.se (corresponding to the manganese dioxide
type), and the other atom in the oxygen molecule is carried to the oxidizable

' Ann. chim. et physiol. (2) 11, 85 (1819).

' P. Raudnitz: Zentr. Physiol. 12, 790 (1899); Z. Biol. 42, 91 (1901).
' George Senter: Z. physikal. Chem. 44, 257 (1903), and 51, 673 (1895); Proc. of the
Roy. Soc. 74, 201 (1894).

* O. Loew: Z. Biol. 43, 256 (1902).

» Philip Shaffer: Am. J. Physiol. 14, 299 (1905).

« Corapt. rend. 124, 1032, 1055 (1897).

ANIMAL OXIDATIONS. 451

substance. Again, the manganese dioxide decomposes with loss of oxygen,
arid in this way the sphtting off of oxygen by the ferment containing the
manganese is effected. Such views are, however, very improbable, because,
according to the researches of Chodat and Bach, the oxidases containing
manganese obtained from plants are inactive in the absence of peroxidases.
The peroxidases themselves have the function of activating the oxidases,
for in the great dilution in which they are present in the juices and cells,
the latter do not readily give up their oxygen from the peroxide formation.
The action of the peroxidases may be compared with the decomposition
of peroxides by means of ferrous salts.

With the help of these hypothetical representations we are able to see
how the cells are not only able to regulate carefully the hydrolytic decom-
positions caused by ferments, but also the oxidations as well. The latter
are directly dependent upon the formation of the peroxidases. Now the
observations upon which this line of reasoning is advanced have been
made upon plants, but there is little doubt that the animal cells conduct
their oxidations in much the same way. All this, however, is purely
hypothetical. It shoidd always be taken into consideration that all
experiments conducted in the study of the action of oxidases have been
with substances which are oxidized without much difficulty. The com-
bustion of such important foodstuffs as albumin, fat, and carbohydrate
is still an obscure process. We do not know definitely in what manner the
oxidizing destruction takes place. It is clear to us, from this discussion,
that the metabolism which takes place within the cells is infinitely com-
plicated, and that in the establishment of the fact that oxygen is taken
up and carbon dioxide eliminated, nothing whatever was determined as to
the real root of the matter. We are now beginning to understand at how
many places the mechanism concerned in the cell-decompositions may be
disturbed and how diverse these disturbances may be.

In the light of our present information concerning the breaking down
and comliustion of the separate foodstuffs in the animal organism, we
may safely assume that the preliminary stage of practically all decompo-
sitions is a hydrolysis. First of all the foodstuffs are subjected to hydro-
lytic cleavage. According to all our present knowledge, amino acids are
formed from albumin, glucose from glycogen, and glycerol and fatty acids
from the fats. We must assume that the other substances which play a
part in the metabolism of the organism are prepared for combustion in an
entirely analogous manner. We know that the nucleins are decomposed
into albumin and nucleic acid, and that these are again broken down into
their simpler components. Purine bodies are thus formed,' which we
believe first lose their nitrogen group and are then prepared for oxidation.
In this case we can establish very accurately the moment at which oxygen

' See Lecture XIII, p. 292.

452 LECTURE XIX.

attacks the molecule. Lecithin is also decomposed into its separate
constituents. In short, an intermediate metabolism takes place in the
tissues, which is caused by processes perfectly similar to those taking place
in the alimentary canal. There the decomposition serves the purpose of
converting substances which are naturally foreign to the organism, but
are contained in the food, into substances which the tissues can assimilate
and incorporate. Hydrolysis is in all cases the preliminary stage to com-
bustion. The fact that oxygen itself in any form is not capal>le of acting
directly upon the cell-nutriment, i.e., that it cannot directly ignite this
fuel, makes it possible for the cells to satisfy the demands for energy within
quite wide limits without regard to external conditions. The cells are
able at all times to utilize certain cleavage-products for building up new
cell-material while they make use of other less valuable substances merely
as fuel. They can adjust their own economy according to their indi-
vidual requirements. The breaking down of the nutriment can take
place from time to time along different lines, as we explained in the case
of sugar. Only at a certain given moment does oxidation ensue. Again,
it is remarkable that, as far as we know at present, we meet with specific
actions. Not every oxidase is capable of oxidizing tyrosine. Here cer-
tain doubts arise as to whether the oxidation takes place exactly as we
have represented, or whether, for example, the oxygen given up by the
activated oxygenase actually of its own accord attacks the difficultly
oxidizable substances without further assistance and consumes it. We
meet with objections to such an assumption, especially as there are many
classes of compounds known of which only one optical isomer is oxidized,
while the other is not. We have already indicated the behavior of the
amino acids. If a rabbit is fed with leucine it is chiefly the /-leucine which
is oxidized, while the greater part of the d-leucine which does not occur in
albumin is eliminated as such.' A great many similar examples are
known. We need merely refer to the observations made with carbo-
hydrates. C. Neuberg and J. Wohlgemuth - injected d-, 1-, and dl-
arabinose into rabbits, and found that 7 . 1 per cent of the /-arabinose, 36
per cent of the d-arabinose, and of the rf?-arabinose 31 per cent of dl-
arabinose plus 9.6 per cent of d-arabinose were eliminated unchanged.
The remander of the material was oxidized. On the other hand, A. Brion '
found that of levo- and mesotartaric acid 93 . 6 to 97 . 3 per cent were oxi-
dized, of racemic acid only 58 . 1 to 75 . 3 per cent, and of de.xtrotartaric
acid 70.7 to 74.4 per cent. We will discuss these facts later. Here they

' J. Wohlgemuth: Ber., 38, 2064 (1905). Schittenhelm and Katzenstein: Z. exper.
Path. Ther. 2, 560 (1906). Abderhalden and Samuely: Z. physiol. Chem. 47, 346
(1906).

« Ber., 34, 1745 (1901), and Z. physiol. Chem. 35, 41 (1902); 37, 530 (1903).

5 Z. physiol. Chem. 25, 283 (1898).

ANIMAL OXIDATIONS. 453

are merely cited to show that the oxidation processes taking place in the
animal organism are by no means to be considered as direct in nature, i.e.,
the cleavage-products as such (the amino acids, dextrose and the fatty
acids), are of themselves not susceptible to oxidation in the tissues under
the prevailing conditions, unless it be assumed that, for example, rf-leucine
does not enter into the metabolism of the cells and does not come in con-
tact with the carriers of oxygen. There is, however, no support to such
an assumption. On the contrary, we know that the muscular cells of the
diabetic are constantly being offered d-glucose. The cells consume albu-
min and fat, or rather their decomposition products, just as well as ever.
Glucose alone they allow to pass on unchanged. The key is lost which
can unlock the energy stored up in the glucose molecule. Now of course
an assumption that the oxydase which is capable of offering oxygen to the
dextrose is absent, helps us here. The fact that the diabetic can complete
the oxidation without difficult}-, if the glucose is previously converted
into a more readily oxidizable form, does not necessarily prove, however,
that the glucose molecule must be opened up in some way before oxida-
tion. It is indeed possible that the glucuronic acid and saccharic acid
offered to the diabetic may be consumed at an entirely different place in
the organism, and not utilized at all for the work of the muscular cells. '
At the same time the empirical knowledge that we possess, indicates that
the foodstuffs as such are not at once suitable for oxidation. Something
must be taken out of the complex molecule before the oxygen in the cells
tissues can act upon it. The investigations of Schittenhelm point in this
direction. Guanine, a cleavage-product of the nucleic acids, is first of all
changed into xanthine:

HN— CO HN— CO HN— C

NHa.C C— NH OC C— NH OC C— NH

\/-.TT I I \/

■CH — > ^CH — *

II II •i!*' \ W ^

N— C— N HN— C— N HN— C— NH

Guanine Xanthine Uric acid

^CO

This transformation takes place with loss of ammonia; a hydrolytic
ferment is active. Now for the first time the molecule is ready for oxida-
tion, and uric acid is formed from it. The latter is then acted upon with
the aid of another ferment in a manner unknown to us. We thus see that
a whole chain of different processes is necessary in order to completely
consume a relatively simple substance, a purine base.

We must regard the breaking down of the amino acids as taking place

» See Lecture XIII, p. 292.

454 LECTURE XIX.

in a quite similar manner. Here, evidently, first of all the NH2 group is
removed by the action of a hydrolytic ferment, and then combustion takes
place.

The fact that we are now getting closer to the i-ealization of the actual
conditions is shown by the recent investigations of Embden, Salomon, and
Schmidt. ' They conducted through the liver of a dog, right after killing
it, blood to which the different amino acids had been added, and found,
for example, that leucine caused a considerable increase of acetone in the
circulation. Shortly previous it was established by Embden and Kal-
berlah ^ that the liver normally produced acetone. Now all of the amino
acids do not yield acetone. Thus, for example, it is not formed from
aminovaleric acid, although a glance at the formulas of these two acids
shows they are closely related compounds:

CH3 CH3
\ /

(r) CH

CH3 CH3

(/?) CH

(/?) CH2

(a) CH.NH2

1

(a) CH.NH2

1

COOH

COOH
Leucine

Aminoisovaleric a

Perhaps the different behavior of these two amino acids gives us an
indication as to the manner in which at least a part of the decomposition
products of albumin is acted upon in the tissues. Embden recalled the
observation of Knoop ' that aromatic fatty acids were decomposed in the
animal body first, so that there was a cleavage in the side-chain between
the a and /? carbon atoms. Thus phenylbutyric acid was first changed
into phenylacetic acid. The latter then appeared as phenaceturic acid in
the urine. It is perfectly possible that the breaking down of the aliphatic
fatty acids takes place in the same way. Thus in the above instance we
can imagine that the leucine and aminovaleric acid were first of all robbed
of the amino group. From the former isobutylacetic acid is formed, and
from the latter isovaleric acid. Then, by loss of carboxyl and oxidation,
isovaleric acid is formed from the isobutylacetic acid, and isobutyric
acid from the isovaleric acid. Next cleavage takes place between the a
and /? carbon atoms. Now if these ideas are correct, we must expect that

' Hofmeister's Beitrage, 8, Heft 3/4 (1906).
' Ibid.

' Der Abbau aromatischer Fettsiiuren im Tierkorper. Habil.-Schrift, Freiburg i. B.
1904.

ANIMAL OXIDATIONS.

455

isobutyl acetic acid itself is not broken down into acetone, while this will
be the case with isovaleric acid, as the following formulas show:

CH3 CH3

u

\ /

'B"T3

c

3 "S

CH3 CH3

1

s

\ /

(/?) CH2

(/?) CH

1

(a) CH2

(a) CH2

COOH

COOH

Isobutylacetic

acid

Isovaleric acid

CH:

CO

CH3

Acetone

Direct experiment confirms this explanation. The decomposition of
leucine, therefore, may be represented as taking place in the following
stages :

CH3 CH3
CH
CHs^

CH

(NH2)

COOH
Leucine

CH3 CH3
\ /
CH

- I
CH2

I
COOH

Isovaleric acid

CH3 CH3

\ /

CO

Acetone

In place of the isovaleric acid, naturally the corresponding aldehyde or
alcohol may appear:

CH3 CH3
\ /
CH

I
CH2

CHO

Isovaleric aldehyde

CH3 CH3
\ /
CH

I
CH2

I

CH2OH
Isoamylalcohol

We have intentionally gone into this hypothesis at length in order to
emphasize the complexity of such a process in contrast to the simple idea
of combustion in the tissues that is generally assumed. Many observa-
tions speak for the above conception of the breaking down of the amino
acids. We have discussed this with tyrosine and phenylalanine. On the
other hand, we must admit that we are not yet able to understand why
the animal organism can split off a carboxyl group from leucine, but not

. 456 LECTURE XIX.

from isobutylacetic acid. There seems to be an intimate relation to the
urea formation. The NH2 and CO groups leave the molecule of the amino
acid at one time. With the proof of the acetone formation from products
obtained from albumin, we obtain for the first time a clear idea concerning
the utilization of the carbon chains free from nitrogen from certain amino
acids. We learn in this way to consider the formation of acetone as a
normal process, it being an intermediate product in the decomposition of
leucine. One source of acetone is thus established. Perhaps from this
stage in the breaking down of the amino acids we can trace their relation
to the carbohydrates and fats.

We do not yet know how the preliminary preparation of the glucose
molecule for oxidation is effected. In the case of the fatty acids, however,
we are justified in assuming that oxidation is preceded as above described
by a cleavage between the a and /? carbon atoms. It seems probable that
the eventual oxidation always takes place with products having but few
carbon atoms in the chain. With these presumptions, we can at least
draw a picture of the oxidation processes in the animal organism, which
will at least not contradict any known facts. The cells prepare substances
for oxidation as they require energy. They cannot effect the oxidation of
d-leucine, for example, because no ferment is present which is capable
of breaking the molecule down sufficiently to make the Sxygen accessible
to it. The cells do their work in stages. At any moment the decom-
position may be stopped, and the products already formed, used for new
syntheses. They do not decompose suddenly. We cannot by any means
compare the oxidation in the animal organism with a conflagration.
Everything is regulated to the most minute detail. Cleavage and oxida-
tion take place alternately, so that the cell can utilize the energy it obtains
from step to step, and only in this way is it possible to regulate so care-
fully the heat supply.

The difficulties which are met with in attempting to explain animal,
as well as vegetable, oxidations, have led to various hypotheses which depend
upon the fact that the difficultly oxidizable substances are made capable
of taking up oxygen directly only by means of some function exerted by
the protoplasm. These attempts at explanation, however, do not rest
upon any experimental basis. They are far in advance of our knowledge
concerning the nature of the cell-protoplasm. It is for this reason that it
is so difficult to submit them to experimental proof. 0. Loew ' traces
the oxidation to the unstalsle condition of the albuminoid in protoplasm.
It transfers the lively movement of the atoms in the active albumin mole-
cule to the oxygen and the oxidizable substance. In this way there is a
loosening up of the molecule, so that the atom of oxygen is offered a point

' Ber., 35, 2487 (1902). — cf. E. Wolff: Die chemische Energie der lebenden Zellen.
Munich, 1899.

ANIMAL OXIDATIONS. 457

of attack. We shall not enter here into a discussion of this or a number of
similar hypotheses. It may be merely said concerning them that they
are not very helpful, because we know practically nothing concerning the
■ chemistry of the protoplasm.

Oxygen has, as we have seen, not only the function of enabling the
organism. to make use of the energy stored up in the decomposition products
of the food, but it also serves frequently to prevent the cells from suffering
injury. It is entirely wrong to assume that oxygen, after it once enters
into reaction, immediately burns up the substance completely. We are
certain that in many cases only a partial oxidation takes place; i.e., the
oxidation takes place in stages. This fact also precludes any simple
explanation of animal or vegetable oxidations. The cell must in each
individual case determine the degree of oxidation. With activating of
the oxygen, or by the carrying of oxygen by means of a peroxide formation,
the course of oxidation in the animal tissues is by no means explained.
Exactly as the chemist may choose special oxidizing agents, and, by estab-
lishing the conditions, he may regulate the degree of oxidation, so in the
same way the cell is capable of regulating the oxidation according to its
requirements. Particularly instructive examples . are shown by the
behavior of certain foreign substances, injurious to the cells, from whose
action the organism protects itself, as we have repeatedly seen, in a num-
ber of different ways. Rudolph Cohn ' has shown that methylquinolin
is for the most part entirely oxidized, and that the same is true of
o-nitrobenzaldehyde." With santonin,' CisHigOs, on the other hand, the
oxidation is not carried so far but it is changed into oxysantonin, C15H1SO4.
The animal organism prepares a large number of such substances by
coupling them with certain substances such as sulphuric acid, glycocoll,
glucuronic acid, and urea. The first two of these substances just named
come from albumin, while glucuronic acid results from carbohydrates.
Sulphuric acid combines with a great many substances of a phenolic
nature; thus, ordinary phenol when introduced into the body leaves it in
the form of potassium phenyl sulphate:*

CeHsOH + HO . SO3K = CeHs . 0 . SO3K + H2O.

In this case, which was first noticed and is the simplest of all, the
coupling takes place directly. Sometimes, however, the phenol is first
oxidized. In this way quinol is formed, which then unites with the
sulphuric acid:'

HO . CgHs -h 0 = HO . C6H4 . OH

HO . C6H4 . OH + HO . SO3K = HO . C6H4 . 0 . SO3K + H2O

' Z. physiol. Chem. 20, 210 (1895).

' Ibid. 17, 274 (159.3).

3 M. Jaff^: ibid. 22, 538 (1896-97).

' Baumann and Herter: ibid. 1, 244 (1877-78).

' Baumann and Preusse: Z. physiol. Chem. 3, 156 (1879).

458 LECTURE XIX.

Here, the objfiot of the oxidation is not so apparent, because the combina-
tion with sulphuric acid takes place just as easily before as a'fter. In other
cases it is necessary for the poisonous substance to be oxidized first, in
order to make it possible for the combination with sulphuric acid to take
place. Thus we know that benzene is fii-st changed into phenol/ indole
to indoxyl, and skatole to skatoxyl.^ Acetanilide appears in the urine as
p-aminophenol, as jj-acetylaminophenol, and as the anhydride of hydroxy-
phenylcarbamic acid partly united with sulphuric acid and in part with
glucuronic acid.^ In the group of glycocoU conjugates we have xylene
converted to toluic acid : *

CH3 . C6H4 . CH3 + 3 0 = CH3 . C6H4 . COOH + H2O.

Mesitylene is changed into mesitylenic acid,^ and cymene to cumic acid."
For conjugation with glucuronic acid, similarly, the cells act upon poi-
sonous su-bstances in various ways. Thus, for example, o-nitrotoluene
is changed to nitrobenzyl alcohol, which then unites with glucuronic acid: '

NO2 . C6H4 . CH3 -h 0 = NO2 . C6H4 . CHoOH
NO2 . C6H4 . CH2OH + C6H10O7 = NO2 . C6H4 . CH2O . CeHgOe + H2O »

We have distinguished in the case of all the organic foodstuffs two
different functions which they have in the cells of the animal organism.
On the one hand they may serve as sources of energy, while on the other
hand they may serve as material for the construction of new tissue. Like-
wise it is possible for the inorganic salts to develop energy by physical
methods; but their chief use, however, is in the formation of new material,
whether it be due to an intimate union with organic material, or whether
the unchanged inorganic salt is in an indispensable constituent of the cells
and that their activity only results from its presence. Of oxygen we
have spoken of but one function, that of making energy available. The
question naturally arises whether oxygen itself is not used as building
material in the construction of new cells? There are, as a matter of fact,
certain observations which seem to indicate that the entrance of oxygen
into the contents of the cell, the protoplasm, plays an important part in the
excital)ility of the cell. Kiihne recognized the fact that the protoplasm
of Ama-bce, Myxomijcetes, and the filament hairs of Tradescantia, lost its

' Schultzen, Naunyn, and Munk: Du Bois' Arch. 1876, 340, and I. Munk: Pfliiger's
Arch. 12, 142 and 148 (1876).

' Baumann and Brieger: Z. physiol. Chera. 3, 254 (1879).

3 Jaff^ and Hilbert: Z. physiol. Chem. 12, 295 (1888). Morner; ibid. 13, 12 (1889).

* Schultzen and Xaunyn: Du Bois' Arch. 1876, 353.
^ Nencki: Arch, exper. Path. Pharm. 1, 420 (1873).

'■ Nencki and Ziegler: Ber. 5, 749 (1872).
' Jaff«: Z. physiol. Chem. 2, 47 (1878-79).

* Cf. Fromm: Die chemischen Schutzmittel des Tierkorpers bei Vergiftungen. Strass-
burg, 1903.

ANIMAL OXIDATIONS. 459

motion and excitability when in hydrogen, but became active when placed
in oxygen again.' Max Verworn ^ confirmed these observations on Rhizo-
poda of the Red Sea. It is difficult to perform such experiments upon
the cells of more highly organized forms, because they contain combined
oxygen, which enables the muscles, for example, to work for some time
with production of carbon dioxide in an atmosphere devoid of oxygen.^
If, however, a muscle is allowed to act until exhausted, it is found that it
becomes active again, only when provided with a new supply of oxygen.
Such relations have been established for the muscles of the heart. Finally,
as is well known, E. Pfluger ^ has shown that frogs which were kept at low
temperatures in pure nitrogen gradually lost their excitability, but regained
it again, even after remaining twenty-four hours in this atmosphere, on
being placed in the air once more. We have to thank Max Verworn ' for
a very interesting experiment in this direction. He replaced all the blood
in a frog with a physiological salt solution, containing 0.6 to 0.8 per cent.
The salt solution was free from oxygen. If now by injection of strychnine
the ganglion cells of the spinal medulla were excited, as much as possible,
then the neurons worked (under the constant streaming of salt solution)
until finally all the oxygen stores had been exhausted. In less than an
hour the reflex excitability is lost. After this the strongest irritation
produces no reflex action. If now a salt solution containing dissolved
oxygen is caused to circulate through the blood-vessels, the frog revives
within a few minutes, and again shows the increased excitability caused by
the strychnine. Every time the circulation of the salt solution containing
the dissolved oxygen is stopped, the ganglion cells at once become unex-
citable. The experiment may be repeated over and over again, with the
same frog. Since in this experiment the ganglion cells are not provided
with fresh nutriment, it is not possible to explain the action of oxygen by
the simple assumption that its absence prevents the complete combustion
of the decomposition products, while at the moment cxygen enters the
energy becomes available to the cells. To be sure, we are quite ignorant
concerning the work of the ganglion cells. We do not know what their
expenditure of energy is. It may be very small. If we remember that
the oxygen of itself is not able to attack the decomposition products from
the food, but requires assistance on the part of the cell before the oxygen
can find a point of attack, then it is not perfectly clear why the oxygen

' Untersuchungen uber das Protoplasma und die Kontraktilitat, Leipsic, 1864. Z.
Biol: 36, 425 (1898).

' Die Bevvegung der lebenden Substanz. Jena, 1892. Cf. also Die Biogenhypothese.
Jena, 1903.

' Kronecker: Ueber die Ermiidung und Erholung der quergestreiften Muskeln
(1871). Joteyko: La fatigue et la respiration 6!^raentaire du muscle. Paris, 1896.

* Pfluger's Arch. 10, 2.51 CiS75).

= Arch. Anat. Physiol. 1900, Suppl. 152.

460 LECTURE XIX.

should nevertheless revive the excitability as shown by the oxidation and
development of energy. It is not necessary in every case that all of the
hydrolytic products should be consumed. The cell is able to be very eco-
nomical with its fuel. On the other hand, it is perfectly possible that
oxygen under these conditions may play an unusual part in the mechanism
of the cell work. We cannot work out such problems successfully until we
have a more accurate insight into the oxidations of the cells and tissues,
and as long as our physical and chemical conceptions of the proto-
plasm are still vague. We meet here with a great many riddles, which
for the present are unanswerable — for the present, we say, because
we can scarcely doubt that before long the rapid progress of biological
chemistry will bring clearness to our conception of these complicated
processes. An advance in the science is only possible, however, when it
is clearly and sharply recognized where the facts end and the hypotheses
begin. We can only build upon the former, and the latter serve merely
as a framework which is of value only when it is possible to replace the
fantasies of the brain little by little with facts verified by experimentation.

LECTURE XX.

FERMENTS.'

We have repeatedly encountered the conception ferment in our discussion
of the transformations of our organic foodstuffs in the ahmentary tract and
in the tissues. We have seen that the proteins are subjected to the action
of pepsin-hydrochloric acid in the stomach, and to trypsin in the intestine,
and that the ferment diastase decomposes almost completely the compli-
cated carbohydrates in the mouth, and to a much greater extent in the
intestine, while the ferment lipase hydrolyzes the fats in the stomach and
in the intestine. The ferments also participate largely in tissue-metabolism.
We meet them wherever life processes occur. They are distributed as
widely in the vegetable world as in the animal kingdom. Fermentation
phenomena are so striking that they were recognized in very early times,
alcoholic fermentation first attracting attention. Spallanzani,^ in 1785,
showed the solvent action of the gastric juice on protein, while Kirchhoff,'
a few years later, called attention to the fact that fresh gluten will saccharify
starch. Liebig and Wohler ^ discovered emulsin, which spHts amygdalin,
and Bussy identified myrosin. If to these we add the discovery of the
oxidases by Schonbein, and the preparation by Berthelot of yeast invertin,
the action of which upon cane-sugar was known even to Mitscherlich, we
shall include practically all of the most important fermentation phenomena
known at the end of the nineteenth century. It is only during the last
few decades that we have really made any great progress in our knowledge
concerning the various kinds of ferments.

Before discussing the nature of the ferments and their action, we must
state in advance that we have not yet succeeded in characterizing the
ferments as chemical individuals. We know practically nothing concern-

* In order to avoid confusion, we shall employ the term "ferment" in its original sense,
— something which causes fermentation. It has been customary to distinguish between
organized ferments and unorganized ferments, or enzymes; but it will be shown in the
following pages that such a distinction was based upon a misapprehension. For a more
complete orientation concerning ferments and fermentations, consult J. Reynolds
Green: The Enzymes; and Carl Oppenheimer: Die Fermente und ihre Wirkungen,
Leipzig, 1903.

2 Lazz. Spallanzani: Versuche iiber d. Verdauungsgeschaft. Leipzig (1785).

3 Schweiger's Journal, 14, 389 (1815). Dubrunfaut: Soc. Agricult. Paris (1823.)

* Poggendorff's Ann. 41, 345 (1837).

461

462 LECTURE XX.

ing their constitution; in fact, we do not even know to what class of com-
pounds they belong, or whether they form a class of their own. It has
always been customary to classify the ferments with the proteins. There
were various reasons for this assumption. Until recently, the composition
and structure of the proteins were as little understood as that of the fer-
ments themselves, while the fats and carbohydrates had been thoroughly
investigated. Our knowledge concerning the compositions of the latter
compounds makes it seem improbable that the ferments belong to either
of these two classes. The proteins, on the other hand, with their compli-
cated structure and their various elementary components, are far more
likely to include the ferments, endowed as they are with such numerous
and finely differentiated functions. We are, however, at present unable
with our limited knowledge of the proteins to draw any conclusions regard-
ing the structure of the ferments. It is often stated, in order to show their
albuminous nature, that the pure ferments give the reactions of the proteins.
Unfortunately, we have not yet succeeded in isolating any ferment in a
pure state. In the first place, we have absolutely no criterion of purity.
Pekelharing ' and M. Nencki and N. Sieber ^ have recently attempted to
purify the pepsin of the stomach, the former claiming to have accomplished
the feat. Until we have some clear conception of the composition of the
ferments, it is of little avail to discuss their nature. We are not even jus-
tified in assuming that the ferments belong to a single class of compounds.
It is possible that the fats and carbohydrates also take part in their com-
position. For the present we are unable to state that the identification
of this or that compound actually indicates that it belongs to the ferment.
Such substance may simply be dragged down mechanically in the process
of isolating the ferment. All of our methods for the preparation of the
ferments are such that this assumption is justified.

The ferments are unquestionably closely related to the life-processes of
the cells. They are to be directly looked upon as their secretion products.
The ferments, until recently, were sharply divided into two groups, the
organized ferments, or simply ferments, and the unorganized ferments, or
enzymes. Those which were only active in the presence of the living cell
were included with the former, while those which acted independently
of the cells, such as diastase, pepsin, trypsin, etc., were placed in the
latter class. The designation organized or unorganized, was to give the
impression that, in the first case, the protoplasm of the cell was absolutely
necessary for performing the function of the ferment, while the unorgan-
ized ferments were capable of accomplishing their work without any
further assistance. It is true, however, that we are not at all acquainted

' Z. physiol. Chem. 22, 233 (1896/97); 35, 8 (1902).
' Ibid. 32, 291 (1901).

FERMENTS. 463

with the ferments as such. We only recognize their presence by their
activity, which alone distinguishes them. This, in principle, is the same
for the organized as the xinorganized ferments. The cell produces fer-
ments, which it requires for its own economy, and others, which it
sends out, to produce results that will directly or indirectly benefit
it. The assumption that the ferments which were active when away
from the cells had other powers than those remaining in the cell was
entirely arbitrary. Especially, as it had never been found possible to
isolate such a ferment, i.e., an organized one, from the cell, and bring
it into activity in its isolated form, there was no logical ground for
sharply differentiating between organized and unorganized ferments. We
know that all ferments are more or less subject to external influences.
Definite conditions must be fulfilled to develop their action. Thus, for
niost ferments the optimum temperature lies between 35° and 4.5° C. The
reaction of the medium in which the development occurs is also of con-
siderable importance. Pepsin, for instance, requires a hydrochloric acid
solution, while trypsin acts in a neutral or faintly alkaline medium. When
we find, furthermore, that the formation of ferments by the cells and their
activation has recently been recognized as a complicated process dependent
upon certain influences, we can readily understand why a definite ferment
ceases to act when it has been torn away from its original sphere of activity.
We do not know how many ferments the individual cell contains. It is very
probable that the ferments are first produced in an inactive form, as zymo-
gens, and only activated by the cells when they are needed. It is also
possible that the individual ferments are very closely related in their work,
i.e., they act together, and assist one another. We also know that many
ferments have their action restricted by the decomposition products which
they themselves produce. Such an accumulation of cleavage-products
would hardly occur in the cell itself, for they are quickly acted upon by
other ferments. If, however, such a cell-ferment were forced to develop
its activity outside of the cell, we can easily see how it might soon cease
to 1)6 efficient.

The discovery by E. Buchner,' that it is possible to isolate and separate
from the cell structure, the ferment from yeast which converts sugar into
carbon dioxide and alcohol, proved for the first time that the similar effects
of ferment and cell activities are evidently due to the fact that the same
agents are at work in each case. This discovery, which does away with
our previous distinction of ferment and enzyme, was carried out in the
following manner: Buchner - ground a kilogram of yeast with a kilogram of
quartz-sand and 2.3 kilograms of infusorial earth. His purpose was to

' E. Buchner, H. Buchner and M. Hahn: Die Zymasegarung. Miinchen & Berlin
(1903).

' Albert and Buchner: Ber. 33, 266, 971 (1900).

464 LECTURE XX.

destroy the cell walls and permit the release of the cell protoplasm. The
whole mass was then subjected to a pressure of from 400 to 500 atmospheres.
The expressed liquor had a slightly acid reaction, it was weakly opalescent,
and contained albuminous material. It contained the active principle
which converts sugar into carbon dioxide and alcohol. Buchner calls it
zymase. It is very unstable, the filtered juice losing its activity after a
few days. The zymase is destroyed at 40-50 degrees. It can be dried,
and in this form keeps much better. It can also be precipitated from its
solution by the addition of alcohol or ether,' by which we obtain a white
powder, only partially soluble in water, but more so in glycerol." The glycerol
extract is very active. Zymase has recently been obtained directly from
the yeast cell, by first killing the cell by contact with ether-alcohol or ether-
acetone. The zymase remains active after this treatment. Such prepa-
rations, which may be kept for a considerable time, are called preserved
yeasts.

A vigorous objection was quickly raised against classifying zj'mase with
the unorganized ferments. Special attention was called to the fact that
the zymase in the expressed liquor was active only for a short time. This
was explained on the assumption that the zymase must be looked upon as
a part of the protoplasm, which, on being separated from the remainder
of the cell contents, possessed only a short period of activity. Quite aside
from the fact that it is now possible to prepare zj^mase with better keep-
ing qualities, this objection is met by what has been said above con-
cerning the cell ferments and their dependence upon their environment
and other ferments, etc. The expressed liquor contains not only zymase,
but also other ferments, of which one, the so-called endotryptase, quickly
destroys zymase. Zymase is, in fact, very susceptible towards proteolytic
ferments. It is also perfectly clear, that those ferments contained within
the cells would naturally be much more susceptible to unusual conditions
than those other ferments which are given off by the cells, and are undoubt-
edly better equipped for battle with the outer world.

That, moreover, fermentation is not necessarily dependent upon living
cells, was already indicated by an observation of Fiechter,' who showed
that hydrocyanic acid completely arrests the vital processes of yeast, but
does not at once stop the fermentation.

The tracing of the conversion of glucose to alcohol to a fermentation
process is not the only case where a so-called " life-process " has been
proved to take place independently of the living organism. Zymase, like
all other ferments, must be regarded as a product resulting from the life-
processes of the cells. The enigma of their formation and existence still

' R. Albert: Ibid. 33, 2775 (1900).
' R. Albert: Ibid. 35, 2375 (1902).
' Wirkung der Blausaure. Diss. Basel (1S75).

FERMENTS. 465

remains even after they have been isolated. ' E. Buchner and J. Meisen-
heimer ' have recently succeeded, by using the acetone method of procedure,
in obtaining a preparation from Bacillus Delbriicki (Leichmann) which
produced lactic acid from grape-sugar in the same manner as the bacillus
itself. They also obtained preserved preparations from beer-acetic-acid
bacteria, using the acetone method, which converted alcohol into acetic
acid.

There is evidentlj' no sharp dividing line between the individual cell-
ferments and the free, unorganized ferments. There are some which are
undoubtedly closely related to the cell contents, and others which are more
loosely united to the cell contents than is the case with yeast zymase, and
can consequently be more easily isolated from the cells. Finally, there
are those ferments which are given off by the cells themselves.

The secretion by the gland cells can be followed directly by histological
methods, and it is quite possible that certain visible changes of the gland
cells may be related to the formation of ferments. The morphological
changes in the cells of the pancreas have been studied in particular.
Although the cells of the resting gland are but slightly distinguishable
from one another, we observe sharp, generally douljle, boundary lines, at
the instant when activitj- begins. The cells, and likewise the gland itself,
change their form. They become filled out. We observe kernels, which
belong to the inner zone of the cells, migrate towards the lumen of the
gland, become smaller, and finally disappear. The cell-changes in the
salivary glands, especially the parotid, have been very carefully studied
during their activity. The cells decrease in size during secretion. The
nucleus, usually angular, becomes rounded, and shows granules very
distinctly. The clear, homogeneous substance, predominating when at
rest, decreases in amount, while the granular substance increases. It is
difficult to say whether the gland cells give up a part of their protoplasm
during the secretion, or if, as seems more probable, products are formed
which then go over into the secretion. It is possible that the granules
mentioned possess some relation to the formation of ferments.

The fact that a great many ferments are not secreted as such, but in an
inactive form, must be looked upon as of the greatest significance for the
conception of fermentation processes. The activation results from the
influence of another substance which is often produced at another place,
and does not necessarily form a part of the ferment, and may even be of
simpler composition. We call the secreted inactive ferment a zymogen or
proferment. Thus, we know that the pepsin zymogen is activated by
hydrochloric acid, while the trypsin zymogen requires the enterokinase.
Undoubtedly there are innumerable other ferments which are secreted in

' Ber. 36, 634 (1903).

466 LECTURE XX.

an inactive form, both in the animal and the vegetable organisms. The
cell is thus enabled to regulate its entire metabolism. It only activates
the ferments when it needs them. The way this activating process is
brought about is still unknown. We can imagine that the activating agent
sphts the zymogen, perhaps forming a smaller molecule, or possibly it
breaks open an anhydride or lactone formation, thus permitting those
groups to act which are able to force an entrance into the material to be
acted upon.

Although the discovery of the wide distribution of ferments and their
action, and the knowledge that the cell-functions in a narrow sense
correspond to such processes, opens up new paths and points of view for
Biology, although on the other hand we must not forget that the great
mystery of cell-life remains unsolved. The living cell produces the fer-
ment; of this there is no doubt. At this point we encounter the most
important problems of the whole subject of biology. We should be mak-
ing a great mistake if we were to say that the knowledge of fermen-
tation reactions has solved the mystery of life. It has, to be sure, cleared
up many processes which were previously obscure, and has given us a
very much clearer conception of the whole subject of metabolism. If,
however, we turn from fermentation to the ferments themselves, we imme-
diately touch the unknown. The ferments point to the cells and their
metabolism and functions. It would be equally short-sighted, and lead to
a complete misunderstanding of biological chemistry, if we were to consider
a solution of this mystery as impossible, and content ourselves with an
undefinable conception of " the vital force." There can be no doubt that
the rapidly advancing biological science will attack the problem of the
chemistry of the ferments as soon as the time is ripe. The mystery of
the ferments will disappear as soon as we are able to replace our conception
with a chemical representation. In the attempt to explain biological
processes more and more in accordance with the laws governing the exact
sciences, it is never advisable to wipe away the boundary between the
knowledge gained by exact methods and what has been established by
mere hypotheses. The more sharply we separate the known from the
unknown, the freer will be the development of our further investi-
gations, and the more independently will the facts, as such, speak for
themselves.

Let us review what we know about the nature of fermentation from this
point of view. As a result of our unfamiliarity with the chemical nature
of the ferments, we are deprived of one of the most important supports
in any exact study concerning them, and are temporarily restricted to
hypotheses in explaining their manner of action. The number of ferments
is very large. We can here only refer to such as are in harmony with experi-
mental facts.

FERMENTS. 467

Let us, at the start, ascertain what are the characteristics of the ferments
themselves.

The first remarkable fact is that they are never found as end-products
of the reactions. They remain unchanged. The smallest amounts
suffice to repeat the same reaction a countless number of times. Thus,
invertase is capable of inverting at least 200,000 times its own weight of
cane-sugar,' rennin at least 400,000 parts of casein.

The action of the ferments should, theoretically, be an unlimited one.
There is, however, as has been shown in the case of rennin,^ a gradual loss
of efficiency. This is not, however, caused by the reaction itself. We also
know, as we shall see more in detail later, that the ferments have a specific
action, i.e., trypsin, for instance, only attacks protein, and never the car-
bohydrates or fats; while diastase never acts on albumin, nor does lipase
ever have any effect upon carbohydrates or albumins. We know, further-
more, that the ferments are produced by living cells, some of them being
given up by the cells, while others are retained in the cell itself. Finally,
we are, in most cases, acquainted with the end-products, and are thus able
to draw conclusions regarding the nature of most reactions which they
cause to take place.

Now there are in inorganic chemistry a great many facts known, which
remind us very much of the behavior of ferments. We refer to those
chemical processes in which the presence of a minimum amount of a
given substance is sufficient to produce a marked acceleration. We
speak in such cases of catalyzers, and call the whole process catalysis, or
one of contact action. Ostwald ^ defines catalysis as an acceleration of a
slow chemical change brought about by the aid of a foreign substance. Accord-
ing to this definition, the reaction in question is not started by the cata-
lyzer, but whereas it takes place very slowly of itself — perhaps even to
an imperceptible extent — the rate of change is accelerated by the presence
of a specific substance. In support of this assumption we have the fact
that increasing the amount of the catalyzer causes further acceleration of
the catalysis. If the process served merely to start the chemical change,
we should hardly expect that the amount of catalyzer present would have
any influence, and at all events the speed of the reaction would be inde-
pendent of the amount of contact substance present. We shall subse-
quently come back to this definition. We are interested here in the simi-
larity between these contact substances and the ferments. One of the
chief common characteristics is the fact that neither appears among the

' J. Chem. Soc. Trans. 57, 8.34 (1890).

' Reichel and Spiro: Hofmeister's Beitrage, 6, 68 (1904), and 7, 479 (1905).

' Grundriss der allgemeinen Chemie, .3d ed. (1889), p. 514. Ueber Katalyse. Lecture.
Hirzel. Leipzig (1902). Compare also, G. Bredig: Die Elemente der chemischen Kinetik,
etc., Ergeb. Physiol. (Asher and Spiro) 1, 134 (1902).

468 LECTURE XX.

end-products of the reaction, and both are effective when present in very
minute quantities. Thus, a very small amount of nitrous acid is sufficient
to convert relatively large quantities of sulphur dioxide and atmospheric
oxygen into sulphuric acid, tij.^tt to ^W.tijsjs milligram of colloidal
platinum or manganese dioxide, or 3.ij(j(y milligram of gold, will cause
the decomposition of more than one million times as much hydrogen
f)eroxide. Ernst ' has shown that -^ milligram of colloidal platinum
will catalyze 50,000 times as much oxyhydrogen gas without losing any
of its efficiency. It is of great significance for the conception of the
action of catalyzers to know that there are substances which have the
opposite effect, and retard reactions which are already in progress. Bredig ^
calls these negative catah/zers. Thus, we know that traces of ethylene,
alcohol, ether, oil of turpentine, and ethyl iodide will tend to prevent the
oxidation of phosphoi'us.' Bigelow* for instance, has shown that the
presence of 0.000,0014 gram of mannitol per cubic centimeter will reduce
the rate of the oxidation of 800 times as much sodium sulphite in aqueous
solution to one-half its former value. On the other hand, we also know of
substances which will directly prevent catalysis. These are known as
anti-catali/zers or paraltjzers. As an example of this may be mentioned,
that 0.000,000,001 gram of hydrocyanic acid per cubic centimeter
will reduce the catalytic effect of 0.000,006 gram of colloidal plati-
num in the decomposition of hydrogen peroxide to one-half its original
value.

Not only do we have the above analogies between the ferments and
catalyzers, but there are other similarities, such as the effect of external
conditions upon their activity, e.g. temperature, etc. Certain laws have
also been worked out showing how the rate of the reaction depends upon
the amount of ferment present and upon the temperature, etc., and these
relations illustrate the analogy between the ferments and the above-
mentioned catalyzers. It would be beyond the scope of these lectures to
dwell longer upon these interesting observations. We can abandon the
subject the more readily because if we were to attempt to apply these
principles to the processes in the animal and vegetable organisms, it would
further our insight into the cell processes but little. We must not be
deceived by the little knowledge we have gained regarding catalysis, for,
generally speaking, we know nothing at all concerning the way in which
the catalyzers act. We know merely that their presence is necessary.
We must admit that the relations between catalyzers and ferments are
to be regarded only as analogies, there being no proof that their actions

' Z. physikal. Chem. 37, 448, 454 (1901).

= Bredig and v. Berneck: Ibid. 31, 324 (1899); Bredig and Ikcda, 37, 1, 63 (1901).

' Centnerszwer: Ibid. 26, 1 (1898).

« Ibici. 26, 493, 503 (1898).

FERMENTS. 469

are identical. Even the application of the definition of catalysis to
fermentation requires reflection. The ferment in such a case would
merely serve to accelerate reactions which were already in progress. We
would have to assume, for example, that albumin would be decomposed
hydrolytically at 37 degrees in an aqueous solution or suspension, although
the hydrolysis proceeds so slowly that we are unable to detect its progress.
The addition of pepsin-hydrochloric acid, or of trypsin, accelerates the
reaction to such an extent that we are able to recognize the hydrolysis in a
short time, on account of the appearance of cleavage-products. We can-
not deny that such an explanation is hardly satisfactory. It is not sus-
ceptible to direct proof, and leads to a forced assumption. It is not at
all clear why pepsin-hydrochloric acid should hydrolyze the albumin
molecule so much less and possibly in an entirely different manner than
tiypsin does, or why the latter should split off definite amino acids so
much quicker than others, and furthermore leave certain complexes en-
tirely unaltered. We know, furthermore, that grape-sugar may be decom-
posed in various ways, which must be regarded as fermentation processes.
In butyric acid fermentation, it yields butyric acid, carbon dioxide, and
hydrogen:

CeHioOe = CiHgOo + 2 CO2 + 2 Hj.

In lactic acid fermentation, it is decomposed as follows:

CfiHiaOe = 2 CsHeOg.

Finally, we know, that zymase splits it into ethyl alcohol and carbon
dioxide:

CeHiaOe = 2 CoHgOH + 2 CO2.

The two latter processes are unquestionably pure fermentations. In these
cases, the ferment not only accelerates reaction already existing, but it
also determines the direction and progress of the same. The fact that the
ferments act in such a specific manner, i.e., only attack substances of
definite configuration, leaving other closely related compounds entirely
untouched, indicates very clearly that we cannot be satisfied with the
definition of Ostwald, at least as far as fermentation processes are con-
cerned. We cannot expect to understand perfectly the action of fer-
ments until we have become better acquainted with their chemical
composition. As long as we deal with them only as conceptions, we can
hardly expect to make any progress regarding the nature of their action.
We do not wish to underestimate the value of physical chemistry in this
direction; we merely wish to differentiate between those facts which we
can consider as proved, out of the numerous investigations of recent years,
and those results which are only based upon hypotheses.

470 LECTURE XX.

One of the most interesting properties of the ferments is their specific
action. The animal, and also the plant organism, as we have mentioned
many times, works almost exclusively with optically active carbon com-
pounds, i.e., with compounds which have at least one asymmetric carbon
atom. The asymmetry of the elementary constituents of the cells begins
at the moment of the assimilation of carbon-dioxide ' by the parts of the
plants containing chromophyll, and is transferred directly by the herbivora,
indirectly by the carnivora, into the animal organism. From a compound
containing only one asymmetric carbon atom we can imagine two optical
isomers and one racemic compound formed by a union of the two.^ Even
Pasteur ^ was acquainted with the fact that if we inoculate a solution of
ammonium tartrate, containing a small amount of nutrient salts, with
traces of Penicillium glaucum, a peculiar change takes place. The solu-
tion, which was at first entirely inactive, becomes optically active during ,
the development of this mold, rotating towards the left. The Isevo-
rotation continues to increase, and only assumes a constant value when
the dextrotartaric acid, the optical isomer of the laevotartaric acid, has
been entirely consumed by the mold. This interesting phenomenon can
be explained on the assumption that the mold evidently only utilizes one
of the optical modifications of tartaric acid, while Icevotartaric acid remains
unchanged. After this observation of Pasteur, which was attril^uted to
the action of an organized ferment, others followed. Thus, by the aid of
Penicillium glaucum, the following optically active forms were obtained from
the racemic compounds: c/-mandelic acid,d-aspartic acid,d-leucine,Z-tartaric
acid, Z-mannonic acid lactone, ^-glutamic acid, and Z-glyceric acid.^

Felix Ehrlich ^ has made an interesting discovery in this direction. He
permitted a pure culture yeast to act upon synthetically prepared, racemic
leucine, in the presence of cane-sugar. After a time a distinct odor of
fusel oil was noticed. Isoamyl alcohol was separated from the liquid by
fractional distillation. All the leucine present was not used up in this pro-
cess, but only the Heucine. The d-leucine could be recovered unchanged
from the liquid. The same experiment, using rf-isoleucine, resulted in the
formation of d-amyl alcohol. The splitting of racemic bodies into the
optically active components by means of lower organisms has become an
important method for the preparation of such compounds, and has become
of great significance in identifying synthetically produced substances
with those which occur naturally. As different organisms decompose
different parts of the racemic bodies, it is possible for us to obtain in this

' Compare Lecture IV, p. 54.

' Compare Lecture II, p. 15.

' Compt. rend. 51, 298 (1860).

' Compare C. Winther: Ber. 28, 3000 (1895).

' Z. Vereines Deut. Zuckerind. 55, 592 (1905).

FERMENTS. 471

way either one of the optical isomers, by selecting the proper organism.
Emil Fischer,' who early recognized the significance of stereo-chemical
influences in biological processes, studied the alcoholic fermentation from
this point of view. He found that, of two isomers, yeast will ferment only
one; in fact, the following:

Ferments Does not Ferment ^
d-Glucose ^Glucose

d-Mannose ?-Mannose

d-Galactose Z-Galactose

rf-Fructose Z-Fructose

The configuration of these compounds has been explained by the
researches of Emil Fischer,^ and it is possible from the structural formulis
at hand to determine the influence of the configuration upon the attack
by yeasts. The formulae of the four fermentable sugars are as follows:

COH COH COH CH2OH

H . C . OH HO . C . H H . C . OH CO

I I I I

HO . C . H HO . C . H HO . C . H HO . C . H

H . C . OH H . C . OH HO . C . H H . C . OH

H . C. OH H . C . OH H . C. OH H . C . OH

CHoOH CH2 . OH CH2 . OH CH2 . OH

rf-Glucose f/-Mannose rf-Galactose rf-Fructose

It is clear from these formulae that d-fructose, d-glucose, and d-mannose
closely resemble one another in their stereo-structure. The OH and
H groups are arranged alike in three of the four asymmetric carbon atoms.
It is interesting to note that these three sugars also possess close chemical
relations, as is indicated by many of their transformations, and especially
by the fact that they go over into one another, simply on heating with
alkali. The d-galactose, in its configuration, does not stand so close to the
sugars mentioned. The same also applies to its behavior. It is more
slowly fermented; in fact, some varieties of yeast, such as Saccharomyces
apiculatus and productivis, do not act upon it at all. All of the other
known aldose and ketose sugars remain unacted upon by yeasts. The

' Z. physiol. Chem. 26, 60 (1898-99).

' Ber. 23, 382, 2620; 25, 1259; 27, 20:31; 27, 2985; 27, 3479; 28, 1429; 27, 2035
(1894).

' Compare Lecture II, p. 17.

472 LECTURE XX.

following formula of a non-fermentable sugar, d-talose, will show what
a slight difference in the configuration in the molecule may prevent the
action of the yeast :

COH

HO

.C.

.i.

1

,H

HO

.H

HO

1

.c,

,H

H

.c

.OH

CHa . OH
f/-Talose

Although, from what was said about zymase, we can conceive of the
selective behavior of the cells of PeniciUium glaucum, yeasts, etc., as due
to the action of ferments, it is nevertheless desirable to perform such
experiments with the individual ferments themselves. In many cases the
cell, with its ferments, may conceal such a specific action on account of
the acting together of ferments of different kinds. Emil Fischer has suc-
ceeded in showing that the specific activity of the cells is dependent upon
the ferments contained therein. If aldoses are heated with very weak,
alcoholic hydrochloric acid, we obtain two isomeric glucosides, which are
designated as the a- and /9-compounds. Emil Fischer has assigned the
following formula to these methyl derivatives of t^-glucose :

H .
H,
10

. C . H /

H

1

H

. C . OH

1

CHo . OH

Only one of these two compounds, in fact the (9-form, is hydrolyzed by
emulsin into methyl alcohol and grape-sugar; the a-form remains unacted
upon by this ferment. On the other hand, ferments obtained from yeast
do not attack the /3-glucoside, but hydrolyze the a-form. The stereo-
isomers of the above compounds, a-methyl-Z-glucoside and ;9-methyl-
i-glucoside, are not attacked by either emulsin or the yeast ferments.

FERMENTS.

473

Moreover, the ferments are so sensitive that they can detect differences
in chemical compounds which are, as far as our present knowledge of
structure and stereo-chemistry shows, perfectly analogous. This is clearly
shown by comparing the a- and ,3-methyl-d-glucosides with the a- and ^-
rtlethylxylosides. *

C^. OCH,

CH3O . C^

HO . C . H

H.C

H . C . OH

HO . C . H

I .
H . C/

H.C. OH

CH2 . OH CH2 . OH

a- and /3-methyl-rf-glucosides

H.C. OCH3 CH3O . C^. H

H.C. 0H\.

I
HO . C . H /

H.C. 0H\
HO . C . H

H.C

H ..C-

CH2 . OH CH2

a- and ;9-methylxylosides

OH

While the former are split by either emulsin or the yeast ferments, the
xylosides are not acted upon by either of them.

Emil Fischer made analogous observations with the polysaccharides.
Some of these are also unfermentable, and their different behavior is
undoubtedly due to differences in the configuration of the molecvdes.'

For investigating the relations of the action of ferments to the con-
figuration of the individual compounds, Emil Fischer by his extensive
syntheses in the protein group has opened up recently a new field which
in its diversity far exceeds the relations with the carbohydrates. This
scientist has, as we have already seen, made the cleavage-products of the
proteins unite together in various anhydride-like combinations. The
number of chains which it is possible to unite in this manner is very large.
The number of possible isomers which can be obtained by using different
amino acids and in different sequence, and also by employing the racemic
amino acids, is greatly increased on account of the fact that all of the known
cleavage-products of albumin, with the exception of glycocoll, contain at

Compare also k. Kalanthar : Z. physiol. Chem. 26, 89 (1898).

474 LECTURE XX.

least one asymmetric carbon atom. Now it was of great interest to trace
the behavior of the sj^nthetic polypeptides in the presence of proteolytic
ferments, in order to ascertain whether the fine distinctions which were
noticed with the carbohydrates would also apply here. The synthetic
polypeptids do, in fact, show a decidedly different behavior toward the
pancreatic juice, as is shown by the following table: '

The Pancreatic Juice

Hydrolyzes; Does not hydrolyze;

*Alanyl-glycine Glycyl-alanine

*Alanyl-alanine Glycyl-glycine

*Alanyl-leucine A Alanyl-leucine B

*Leucyl-isoserine A Leucyl-alanine

Glycyl-Z-tyrosine ■ Leucyl-glycine

Leucyl-Z-tyrosine Leucyl-leucine

*Alanyl-glycyl-glycine Aminobutyryl-glycine

*Leueyl-glycyl-glycine Aminobutyryl-aminoljutyrlc acid A

*Glycyl-leucyl-alanine Aminobutyryl-aminobutyric acid B

*Alanyl-leucyl-glycine Aminoisovaleryl-glycine

Dialanyl-cystine Glycyl-phenylalanine

Dileucyl-cystine Leucyl-proline

Tetraglycyl-glycine Diglycyl-glycine

Triglycyl-glycine-ester Triglycyl-glycine

( = Curtius' Biuret-base.) Dileucyl-glycyl-glycine

If we examine these two columns, we observe that the pancreatic fer-
ment has various points of attack. In the first place, the structure of the
individual compounds must be taken into consideration. A good example
here is the behavior of alanyl-glycine:

CH3 . CHCNHa) . CO . NH . CHg . COOH,

and its isomer, glycyl-alanine:

NH2 . CH2 . CO . NH . CH(CH3) . COOH.

The former is hydrolyzed, the latter is not. The nature of the individual
amino acid is also important. Thus, those dipeptides are susceptible to
hydrolysis in which the alanine acts as acyl, examples. being: alanyl-
glycine, alanyl-alanine, alanyl-leucine. The hydroxy acids, tyrosine and
isoserine, have a similar effect when they are at the end of the chain. It is

' Emil Fischer and Emil Abderhalden: Z. physiol. Chem. 46, 52 (1905). Compare
also, Sitzber. Kgl. Preuss. Akad. Wiss. 10 (1905), and Emil Fischer and Peter Bergell:
Ber. 36, 2592 (1903); 37, 3103 (1904).

FERMENTS. 475

possible that the electro-negative character of these compouncfe plays a
part. It has not yet been decided whether the easy cleavage of the two
cystine derivatives is due to this cause, or that the length of the chain is a
factor. It is very noteworthy that the dipeptides, which contain a-amino-
butj'ric acid, and leucine as acyl, offer great resistance.

The influence of configuration is also clearly indicated in this case. The
polypeptides in the above table which are designated by an asterisk, are
racemic compounds. In all of these cases, the hydrolysis is an asym-
metric one, i.e., only half of the racemic body is attacked. The products
resulting from the hydrolysis were the same as those active amino acids
which are contained in the natural protein-substances. A special case is
shown by the contrast between the alanyl-leucine A. and alanyl-leucine B.
In these two racemic compounds are present all four combinations of the
four active amino acids. One racemic compound is d-alflnyl-c?-leucine -|-
Z-alanyl-Meucine; the other, d-alan^d-Z-leucine + Z-alanyl-c?-leucine. Of
these four combinations, the pancreatic juice will attack only that corre-
sponding to rf-alanjd-Z-leucine. This fact is of great significance. It
supplies us with a means for determining directly the configuration of the
synthetic polypeptides.

The number of the amino acids contained in the complex molecule is
also of influence. The glycine chains give us a distinct example of this.
Glycyl-glj'cine, diglycyl-glycine, and triglycyl-glycine are not hydrolyzed,
while tetra-gh^cyl-glycine is acted upon. Leucyl-glycine, also, is not
decomposed, although leucyl-glycyl-glycine is. The reason that dileucyl-
glyc3d-gl}^cine is not decomposed, lies probably in the configuration of the
dileucyl group.

If the cleavage takes place along asymmetric lines, the beginning of
hydrolysis in the previousl}- inactive digesting liquid is established by the
appearance of optical activit}'.

We may here include the observation of 0. Warburg/ who showed that
the ester of racemic leucine is saponified asj^mmetrically bj^ the pancreatic
juice. We do not know what ferment produces this result. Lipase may
be the active principle, as is suggested by an analogous observation of
H. D. Dakin.2

Closely related to these discoveries, is the fact previously mentioned,^
that the animal organism utilizes only one-half of certain racemic sub-
stances, the other optical isomer being eliminated unchanged. These
discoveries show very clearly the similarity of " organized " and unor-
ganized ferments, and justify us in concluding that all ferments must be
considered from the same point of view.

' Ber. 38, 1S7 (1905).

' Proc. Chem. Soc. 19, 161 (1903); J. Physiol. 32, 199 (1905).

' Compare Lecture XIX, p. 452.

476 LECTURE XX.

The fact that the ferments act asymmetrically leads to the assumption
that they themselves are asymmetrically constituted. The ferment must
be exactly fitted to act on the compound which is to uncfergo cleavage.
Possibly the assumption, so often made, that the ferment temporarily
combines with the substance to be hydrolyzed, will account for the specific
behavior of every individual ferment. If this be true, we can easily
understand that only a definite ferment can act upon a given compound.
This assumption is supported by the observation, that pepsin and papain,
for example, form such strong combinations with fibrin that they cannot
be removed by washing. It has also been found that the inversion of
cane-sugar by ferments is, as a rule, the same during equal intervals
of time.

Further light has been thrown upon this problem by tracing the optical
behavior of solutions containing optically-active polypeptides after the
addition of a solution containing ferments.' All results indicate that the
ferment temporarily unites with the substance which it splits. It is
important that the cleavage products tend to prevent the further cleavage
of the substance, in accordance with the mass-action law.

A peculiar significance of the ferments has recently been indicated by
certain observations. Morgenroth ^ found that, after subcutaneous
injection of rennin in small doses, the serum of the animal so treated,
contained a substance which prevented the curdling of milk. This phe-
nomenon is analogous to the production of anti-toxin by the animal
organism after the injection of a toxin. In one case we obtain an anti-
toxin, in the other an anti-rennin. Two per cent of the strongest immu-
nizing-serum which Morgenroth obtained, added to milk, prevented its
curdling even when the ferment was present to the extent of 1 : 20,000.
Without the addition of anti-rennin, the curdling took place when the
ratio was as low as 1 : .3,000,000. Even normal serum is supposed to con-
tain some anti-rennin. Such anii-jerments have also been prepared which
act against pepsin, trypsin, fibrin-ferment, tyrosinase, lactase, and urease.
These experiments are of great importance in two directions if they can be
confirmed. In the first place, this discovery will serve to unite a purely
biological process with another which, up to the present time, has not
been studied by itself. The acquirement of immunity and the formation
of anti-ferments may prove to be analogous phenomena; and the toxins,
which resemble the ferments in many respects, may belong to the same
class of'substances. A further analogy lies in the fact that the ferments

' Abderhalden and Koelker: Z. physiol. Chem. 51, 294 (1907); Abderhalden and
Michaelis: ihid. 52, .326 (1907); Abderhalden and Gigon: ibid. 53, 251 (1907); Abder- ■
halden and Koelker; ibid. 54, 36.3 (1908).

2 Zentr. Bact. 26, 349 (1889); 27, 721 (1900).

FERMENTS. 477

have a toxic effect when injected subcutaneously. Hildebrandt' found
that the lethal dose for a medium-sized rabbit was 0 . 1 gram for pepsin,
invertase, and diastase; 0.05 gram for emulsin and myrosin; and 2 grams
for rennin. All of the injected ferments caused rise of temperature.
Dogs, which had had ferments injected, would not eat, showed thirst, trem-
bling, restlessness, an unsteady gait, and eventually coma. Rabbits
showed principally emaciation, weakness, and sometimes extensor con-
vulsions. Such observations necessarily have only a relative value,
owing to the fact that the nature of the ferments is still unknown, so that
their purity cannot be estimated.

On the other hand, the formation of the anti-ferments and the observed
normal occurrence of these, gives us an indication of the role which, for
example, the ferments absorbed by the intestines perform in their circu-
lation through the bod3^ We can easily imagine that the organism par-
alyzes the activity of the absorbed ferments by the production of the anti-
ferments. The presence of such substances also suggests an explanation
of how the living tissue is protected against self-digestion.^ We can also
imagine that this protection is obtained by the cells altering the material
which they require for constructive purposes, and do not wish to consume,
so that the ferments are unable to find any point of attack. It is certainly
not without significance that the connective tissue, e.g., elastin and
substances like spongin and silk-fibroin, which are not considered as
nutrient materials, contain in large quantities just those amino acids
which make hydrolysis difficult. The substances are, as a matter of
fact, hardly attacked by pepsin-hydrochloric acid or by trypsin. The
cell has only to cause a slight rearrangement of the atoms in the mole-
cules of the assimilated products to form modifications which the fer-
ments are not able to attack, or it may cause them to combine with other
cell-components.

An unsolved problem is the origin of the ferments. It has often been
suggested that they bear a definite relation to the food. In fact, many
observations indicate a close connection between the production of fer-
ments and the assimilation of the food. We merely do not understand
the more intimate relations existing between the two processes. Brown
and Morris ^ have shown that the leaves of many plants contain the most
diastase in the morning, the amount decreasing during the day. If the
assimilation is carried out in the sunlight, the formation of diastase ceases

■ Virchow's Arch. 121, 1 (1890); 131, 26 (189.3).

' E. Weinland, Z. Biol. 43, 86 (1902"), h.is found such anti-fennents in cell-free
extracts of parasitical worms. Extracts from the mucous membranes of the stomach
and intestine, as well as erythrozytes, are credited with retarding the solution of fibrin.
Z. Biol. 41, 1, 146 (1902).

3 J. Chem. Soc. 62, 604 (189.3); 57, 493 (1S90).

478 LECTURE XX.

entirely. The dormant seeds do not contain any ferments, i.e., not in an
active form; tliey do not become active until the beginning of germina-
tion. The barley embryo does not produce diastase when absorbable sugar
is available for it.

The formation of ferments among the molds is likewise influenced by
the nature of the nourishment. If they are provided with albumin, they
will produce proteolytic ferments; if they are cultivated upon starch, they
will form diastase. Furthermore, it is known that yeast, for example,
which has been cultivated for a long time on a specific substratum, can
be " taught " to utilize definite compounds if we gradually withdraw the
other nutriment.

Closely related to these observations is the fact that the plant cells, in
the presence of definite products, also produce the ferments which will
decompose them. This is usually true of the glucoside-splitting ferments.
Thus we find amygdalin together with the ferment emulsin in bitter
almonds; while the glucoside, sinigrin, is accompanied by the ferment
myrosin in black pepper.

There are numerous analogous observations in the animal kingdom. It
was known to Claude Bernard ' that the larvse of Musca lucilia, a kind of
fly, possessed large stores of glycogen, but did not produce any diastase.
The latter appears only when these stores are required by the pupa.

Numerous relations between the kind of food and the amount and
nature of the secreted digestive fluids have become known by the extensive
researches of Pawlow. Nervous influences dominate the production of the
ferments. This was evident long ago from the investigations of Claude
Bernard on the decomposition of glycogen in the liver. Pawlow has
studied this subject more carefully, as we shall see later.

From all of these statements, we involuntarily obtain the impression
that fermentation processes play a large part in the whole economy of the
individual cell, the tissues, and finally the entire organism. This view is
supported by the common occurrence of such processes throughout the
whole plant and animal kingdoms. The fact that their activity begins as
soon as the life proce.sses start indicates their importance. The ferments
are found in very early stages of human and animal embryos. Langen-
dorff ^ detected trypsin especially early. Pepsin is entirely absent among
the carnivora just after birth, but is found in the herbivora. Diastase is
absent from human beings and rabbits previous to birth. The ferments,
evidently, play a part not only in physiological relations, but also in patho-
logical ones. Thus, we observe that fibrin from the bronchial tubes
during croupous-pneumonia is gradually dissolved and finally disappears.'

' Revue scientifique (1873), p. 515.

' Arch. Anat. Physiol. 1879, 95.

* F. Miiller: Verhandl. XX. Kongresses in. Med. zu Weisbaden (1902).

FERMENTS. 479

In this case, apparently the migrant leucocytes which are present in large
numbers furnish the ferment and cause a normal digestion in the lungs.
Some insight into the cell processes, and the ferments which come into
action thereby, was believed to be obtained from Salkowski's discovery
that organs, even when kept perfectly sterile, gradually dissolve of them-
selves.' Cleavage-products are formed at the same time which suggest
the presence of trypsin-like ferments. To be sure, it is generally stated
that the disintegration of the cell contents and especially of the albumin,
in this process which is known as autolysis, is not the same as that which
takes place in a true trypsin digestion. It is true that the same end-
products (amino acids, purine bases, etc.) are formed, but we do not know
whether the decomposition takes place in the same way, and with the
formation of the same intermediate products. It is questionable whether
we are justified at present in making deductions regarding normal cell
metabolism from the autolytic processes which result several days after
death.2

Thus far we have considered cell-metabolisra and fermentation from
only one point of view. We have mentioned only the splitting ferments.
Now we know that extensive syntheses take place in the animal organism.
Since Wohler, in 1824, proved that benzoic acid administered to the animal
organism was neither consumed nor excreted as such, but that a nitro-
genous acid, richer in carbon, appeared in the urine, namely hippuric acid,
which is composed of glycocoll and benzoic acid, a great many other syn-
theses have been proved to take place. We need onlj' to recall the fact
that fats are split into glycerol and fatty acid in the alimentary tract,
only to reappear on the other side of the intestine as fats, and also that the
albumins and complicated carbohydrates are disintegrated into simple
components only to be reconstructed, to realize that synthesis is another
established function of the animal cell. Although, as far as our present
knowledge shows, these syntheses are for the most part simple ones, and
usually consist of the union of two or more molecules with elimination of
water, we must not conclude that the animal cell is incapable of effecting
complicated syntheses. Certain recent results lead us to suspect that the
animal organism is capable of building up complicated structures. The
synthetic processes of the plant and animal organisms were for a long time
hidden in obscurity. Indeed, on purely theoretical grounds, by comparing
ferments with catalyzers, the conclusion was drawn that fermentations
must be reversible processes, and that they may be endothermic as well as
exothermic reactions. As a matter of fact, a whole series of syntheses has
been carried out by the aid of ferments. Thus we may refer to the for-
mation of isomaltose from concentrated d-glucose solutions bv means of

' Z. Klin. Med. 17, Suppl. 77 (1890).
' a. Lecture XII, p. 265.

480 LECTURE XX.

yeast maltase, and to the production of isolactose from glucose and galac-
tose by kephir-lactase, and to the synthesis of amygdalin from mandelo-
nitrile glueoside and grape-sugar with the aid of yeast maltase.' From these
observations there can be no doubt that fermentations are reversible pro-
cesses. This does not prove, however, that the conditions in the tissues and
cells are such that the known ferments are active in this direction to any
great extent. It is very tempting to refer all metabolic processes to fer-
mentation. At one place there is a decomposition, at another, construction,
according to the requirements of the cells. The ferment acts as an inter-
mediary. Many enigmas would thus be solved at one stroke, and many
apparently different processes referred to one simple basis. It is certainly
possible that such a significance really belongs to the ferments, and that
they dominate the entire metabolism. We must, however, confine our-
selves to the facts, and build on them alone as foundation. We are, in the
first place, impressed with the fact" that all the fermentation syntheses so
far carried out satisfactorily do not give rise to products to which the fer-
ment is accustomed, with the po.ssible exception of the amygdalin synthesis.
We do not obtain maltose from grape-sugar, but an isomer, isomaltose;
nor do we obtain lactose from glucose and galactose, but only isolactose.
These facts are certainly not without significance. Can the maltase again
decompose isomaltose, or kephir-lactase hydrolyze isolactose? These
were unsolved problems until recently. Thanks are due E. F. Armstrong ^
for thoroughly studying these fermentation syntheses, and especially
the formation of maltose and isomaltose. Armstrong started with the
fact which we have not previously mentioned, that d-glucose can exist in
stereo-isomeric forms. If we crystallize grape-sugar from alcohol, we only
obtain the a-form. If this is kept for several days at 105 degrees, it goes
over into the /3 form. The two modifications have different optical
behaviors. It has been attempted to represent this type of stereo-isomer-
ism by means of formulae, although a satisfactory explanation is still lack-
ing. If glucose is dissolved in methyl alcohol, containing hydrochloric
acid, glucosides corresponding to both forms are produced. As we have
already seen, only one of these varieties is split by maltase, the other only
by emulsin. The variety hydrolyzed by maltase is the a form. If we
apply these observations to maltose and isomaltose, we can regard the
former as glucose-a-glucoside, and the latter as glucose-/3-glucoside. In
this case also, the maltase is only capable of splitting the a form, while
the ^-glueoside remains unattacked. Now maltase produces synthetically
/?-glucoside, i.e., the glueoside which it cannot decompose. The following
experiments completely cleared up these relations. If glucose was treated

' Cf. Lecture III, pp. 37, .38.

' Pro. Roy. Soc. 76 (B), 592 (1905); 19, 209 (1903); Jour. Chem. See. 83, 1305
(1903).

FERMENTS. 481

with concentrated hydrochloric acid, and hydrochloric gas acid passed
into the liquid at 0 degrees, it was shown, after long standing at 10
degrees, that the fluid contained both glucosides, maltose and isomaltose,
as well as unchanged glucose. The hydrochloric acid makes no distinction;
it works with the a as well as the ^ form of glucose. That both bioses
had, in fact, been formed, was proved by the circumstance that maltase
as well as emulsin produced glucose from it. If glucose was kept at 25
degrees for two or three months in the presence of yeast maltase, it was
shown after removing the unchanged glucose by means of Saccharomyces
intermedians, that isomaltose was present. Emulsin produced rf-glucose
from it, but maltase was unable to do so. When glucose, on the other
hand, was acted upon by emulsin, maltose was produced. These relations
may be summarized as follows:

a-glueose ) synthesized by emulsin.

i
glucose-a-glucoside (maltose) 1 J

j I hydrolyzed by maltase.

a-glucose I

^-glucose 1

I I synthesized by maltase.

glucose- /9-glucoside ,(iso-maltose) I

I \ hydrolyzed by emulsin.

/?-glucose J

Each of these ferments, emulsin and maltase, builds up that biose which
it cannot itself decompose. This synthesis by ferments is, therefore,
different as far as our present knowledge goes from that produced by
true catalyzer.

We have gone into these relations in detail in order to show that we are
at present not justified in concluding that a single ferment in the cells can
effect decomposition or synthesis according to the outer conditions. We
have no reason for believing this. This does not, of course, exclude the
possibility that these processes may be carried out differently in the cells
and tissues. We are, however, not justified in regarding all processes of
metabolism in the tissues and cells as being due to fermentation. It is
absolutely necessary right at this point to confine ourselves to the facts,
and not follow a plausible hypothesis, the value of which is especially
problematical here, for there is a vast amoupt of research to be made.
Although our knowledge of fermentation reactions constantly broadens,
the great mystery regarding the origin and formation of the ferments
remains, and the important question relating to their chemical structure
is still unsolved.

Let us now turn to the classification of the ferments. We have often

482 LECTURE XX.

encountered them in tracing the course of the organic nutrient materials
in their passage from the alimentary canal to the tissues. It remains
only to classify them in a comprehensive manner. We may divide the
ferments into two main groups: (1) the hydrolytic, and (2) the oxidizing
ferments. The former may be further subdivided according to the material
to be attacked, e.g. (a) ferments which effect the decomposition of the
carbohydrates, (6) the proteolytic ferments which act upon the proteins,
and (c) the fat-splitting ferments. The diastatic ferments belong to class
(a). They hydrolyze starch into dextrins and maltose. The decompo-
sition of maltose into two molecules of dextrose is effected by maltase.
Invertase splits cane-sugar into one molecule of dextrose (rf-glucose) and
one of IsBvulose ((Z-f ructose) . To this group belong a series of ferments
whose characteristics have been less carefully studied, such as cellulase,
which is supposed to decompose cellulose; inulinase, which acts on inu-
lin; seminase, which disintegrates mannans and galactans; and finally
pcctinase, which is responsible for the hydrolysis of pectin. We might
also mention trehalase, mclibiase, and lactase, which decompose the sugars
corresponding to their names. Special ferments are also known which
hydrolyze the glucosides, as well as a urease that changes urea into
ammonium carbonate. To class (6), the proteolytic ferments, belong
pepsin, trypsin, rennin, the fibrin-ferment, and pectase which hydrolyzes
pectin substances. The fat-splitting ferments form a class by them-
selves. Then there is lactic-acid ferment, which produces lactic acid
from sugar. It attacks all of the simple hexoses, and some pentoses, but
not cane-sugar or milk-sugar. It produces chiefly the a-hydroxypropionic
acid, CH3 . CHOH . COOH. It is still a question whether the lactic
acid fermentation is to be looked upon as an independent process.

The second main group comprises the oxidizing ferments which have
already been discussed. They obtain their oxygen either from the air or
from decomposed hydrogen peroxide. To the former class belong the
true oxidases, acting as oxygen carriers to the cells and tissues. Atmos-
pheric oxygen is also utilized during the acetic acid fermentation of
ethyl alcohol.

Alcoholic fermentation is quite a special process. It is a complicated
affair, and the ferments producing it cannot, at present, be assigned to
any of the above groups. As we have already shown,' alcoholic fermenta-
tion plays a part of which we cannot at present estimate the extent in
plant and animal ti.ssues, and inasmuch as an inspection of this process
will give us some idea of the progress of a fermentation reaction, we will
briefly discuss it. The reaction takes place according to the following
general equation:

CeHisOe = 2 C2H5OH + 2 CO2.

■ Cf. Lecture IV, p. 74.

FERMENTS.

483

Until recently the whole process of alcoholic fermentation was sur-
rounded with great obscurity. E. Buchner and J. Meisenheimer ' have
succeeded at least in indicating the way in which the decomposi-
tion proceeds. Thus, in their cell-free fermentations, they invariably
found inactive lactic acid. They concluded that this must be a normal
intermediate product, and formulated the alcoholic fermentation to take
place as follows:

CHO

OH

COOH

COOH

OH

CO2

CHOH

OH

H
H

CH.OH

1

CH.OH

1

H

>

CH2OH

CHOH

CH2 H

CH3

CHs

1

-

> 1 1

CHOH

OH

CO OH

COOH

OH

,

1

OH

1

1

H

CO2

CHOH

1

H

CH . OH

1

CH . OH

1

>

CH2OH

CH2OH

H

CH3

CH3

CH3

Glucose

Hypothetical

2 molecules

Ethyl alcohol

intermediate product

lactic acid

-f CO2

+ 5H2O

At all events, alcoholic fermentation is a very complicated process,
and the question has now arisen, whether it is to be looked upon as brought
about by one or by several ferments. One ferment may convert the sugar
into lactic acid, while a second transforms this into alcohol. There are
also by-products in the alcoholic fermentations. Glycerol, succinic acid,
and acetic acid have been noticed among these. If we employ zymase,
such products are not formed to any extent. They are evidently not a
part of the alcoholic fermentation itself, but are due to other metabolic
changes of the yeast.^

' Ber. 37, 417 (1904).

' For the older conception of alcoholic fermentation, cf. C. v. Nageli; Theorie der
Gahrung. Miinchen (1879).

LECTURE XXI.

THE FUNCTIONS OF. THE DIGESTIVE ORGANS. '

I.

We have up to now considered the transformation of each separate
substance in the alimentary canal by itself, as well as its absorption, assimi-
lation, and subsequent destination until the final products of metabolism
were reached. Such a method of presentation has the advantage that it
gives us a clear idea concerning the behavior of any given foodstuff in
the animal organism, and makes it easier for us to trace the relations of
the separate organs to the remaining groups of foodstuffs and to their
functions. On the other hand, in order to avoid repetition, it was necessary
for us to touch only briefly upon certain very essential points, and, further-
more, there were certain important observations which we could not dis-
cuss at all. We shall now in the following lectures consider each individual
organ of the animal organism by itself, and in this way find opportunity
to mention what we have omitted, and, at the same time, to bind together
certain apparently isolated facts with other analogous ones to a single unit.
It is extremely difficult, and in fact impossible, to draw a sharp line between
pure physiology and physiological chemistry. The time has long since
passed when the latter branch of biological science could be considered as
concerned chiefly with the investigation of the composition of the separate
organs, the fluids of the body, and the excretions and secretions. It has
been recognized for some time that the tracing of the relations of the differ-
ent groups of foodstuffs to one another and their transformations in the
organism, has introduced new problems into the field of physiological
chemistry. With the solution of these problems, which are fundamentally
important for the understanding of the entire metabolism, the limits of the
working field of the physiological chemist are by no means reached, as we
shall see. Physiological chemistry takes part more and more in explaining
the functions of the various organs. Certain of the apparently-very-com-
plicated processes have been brought nearer to our comprehension by the
more recent investigations; and for some functions of which it was not
supposed that there was any relation to chemical processes, newer obser-
vations have opened up entirely new perspectives, especially from a chem-
ical point of view. Undoubtedly physiological chemistry must become
more closely united with pure physiology in order that the fruits obtained
in both fields may become fully ripe.

484

THE FUiNTTIONS OF THE DIGESTIVE ORGANS. 485

Such a point of view is particularly advantageous now that we come to
consider the functions of the various organs in the digestive canal. They
are of most diverse nature according to the part of the digestive stratum
in which they are found. Let us begin with the functions of the upper end
of the alimentary canal, the mouth. In it the food which has been pre-
pared in various ways, is chewed up into small pieces and intimately mixed
with the saliva. In this way the morsels are prepared for digestion. The
saliva comes from the salivary glands and from the mucous membrane of
the mouth. There are three pairs of the former, which may be dis-
tinguished chiefly by the nature of the fluids they secrete. The parotid
(near the ear) gland produces a thin watery fluid containing chiefly albumin
and salts. It is spoken of in general as an albuminous gland. Small glands
of this nature are found as well in the mucous membrane of the nose and
mouth. The other glands are the so-called mucous glands. They, in con-
trast to the former, furnish a glairj^ or more ropy secretion, due to the
mucin which it contains. To this class of glands belong, in the case of most
animals, the submaxillary and sublingual glands. Small mucous glands
are found distributed in the mouth, throat, and cesophagus. This distinc-
tion between two kinds of glands is not a sharp one. Thus the lower jaw
of man contains glands which yield a thin secretion rich in albumin as well
as one of a more mucous nature.

The secretion from each separate gland may be examined by construct-
ing fistulas in the exit ducts, or more simply by introducing a canula into
the mouth of the duct from where it discharges outward. Normally a
mixture of the secretions from all of the glands comes into action. The
extent to which the different glands take part in the formation of the
saliva varies. The secretion of the glands is dependent upon quite a
number of outside influences, as J. P. Pawlow ' has quite recently called
to our attention. In the exercise of their function, they are dependent
upon nervous influences. The innervation of each gland is twofold.
Cerebral and sympathetic fibers lead to each. The submaxillary gland
contains fibers from the chorda tijmpani, and, on the other hand, fibers
enter the blood-vessels of the gland from the si/mpathetic system. This
double innervation also corresponds to the nature of the secretion.
By stimulating the cerebral fibres, in other words, those of the chorda
tympani, an abundant secretion of a thin liquid is produced; whereas, by
stimulating the sympathetic nerve, onl}' a few drops of a viscid liquid rich
in mucin are obtained. The sublingual gland behaves quite similarly. The
parotid gland is also partly dependent upon the cerebral nervous system
(in this case the glossopharyngeal nerve) and partly on the sympathetic.

For some time it was believed that the influence of the nerves which
reach these glands could be explained by an action upon the blood-vessels.

' Ergeb. Physiol. (Asher and Spiro) Jahrg. III., Al)t. I, p. 177 (1904).

486 LECTURE XXI.

If the lingual nerve, which carries the cerebral fibers to the sub-
maxillary gland, is cut, and then the peripheral stump stimulated, the
blood-vessels in the gland become dilated. The blood streams from the
veins with a bright red color similar to that of the arterial blood. At the
same time there is an increased secretion of saliva. On the other hand,
by stimulating the fibers of the sympatheticus, the blood-vessels are con-
tracted. The blood passes more slowly, and the flow from the veins is
small in amount and of a dark blue color. The activity of the secretion
is diminished.' That, on the other hand, the innervation of the blood-
vessels is not the sole function of the above-mentioned nerves, is shown
by the experiments of Heidenhain.- He found that with the lingual nerve
two kinds of nerve fibers enter the gland. If a dog was poisoned with
atropin, then stimulation of the facial nerve fibers caused as before an
acceleration of the blood-stream, while, on the other hand, there was no
increase in the secretion of saliva. From this it is clear that the facial
nerve, as well as the sympathetic, carries fibers to the submaxillary gland
which have a specific action upon the individual cells of the gland. For
the present we know but little concerning the details by which this stim-
ulation is effected. It has not yet been found possible to establish
beyond reasonable doubt the anatomical relations of the nerve fibers to
the cells of the gland.

C. Ludwig and A. Spiess ' have shown that with the activity of the cells
the temperature also rises. They introduced a thermometer in the large
vein of the submaxillary gland of a dog, a second in the exit duct from this
gland, and a third in the carotid artery. If now the facial fibers were
stimulated, then from the time of beginning of the activity of the glands
the thermometers in the saliva and in the vein registered higher tempera-
tures than that of the carotid.

It was at one time believed that the formation of the saliva could be
regarded as a filtration process. It was soon found, however, that the
separation of this liquid was evidently due to a specific activity on the part
of the gland-cells. Even the chemical composition of the saliva indicates
this. It is entirely different from the blood and lymph, and must have been
formed only by means of a specific choice of the individual constituents
of these liquids on the part of the gland-cells. In fact, it is even necessary
for these last to produce for themselves certain substances. Here, we
cannot treat in detail of all the evidence which has been brought forward
against the assumption that the separating of the saHva is a result of a
filtration process. We can only briefly touch upon it. Thus it has

' Claude Bernard: Compt. rend. 47, 245 (1858).

' Pfluger's .\rch. 5, 309 (1872); 17 (1878). See also Barbera: Bull, scienze med.
di Bologna (8), 2, 1 (1902).

3 Sitzber. Wiener Akad. 25, 548 (1857).

THE FUNCTIONS OF THE DIGESTIVE ORGANS. 487

been found that if a mercury manometer be introduced into the exit duct,
then after stimulating the cerebral secretory nerves, the mercury will in a
short time rise 100 miUimeters or more higher in this manometer than in
another one that is placed in the carotid artery. There is, therefore, a
considerable increase of pressure during the secretion of saliva.' It is also
an important fact that stimulation of the secretory nerves has an effect
even in animals from which the blood has been removed completely.
That the cells of the gland are active during the preparation of saliva, is
evident from a microscopic examination during a period of rest and one
of action.^ We have to thank Heidenhain' for this interesting information.
He described the contents of the albuminous glands at rest and after
secretion had taken place, using alcoholic-carmine preparations. In the
first instance, a shrunken, finely granular substance is seen in a clear,
uncolored background. The nucleus itself appears as an irregular, serrated
structure without any distinct nucleolus. On making preparations in the
same way of glands which have been in marked activity for some
time, under nervous stimulation, there is a quite different appear-
ance. The size of the cells has increased to a greater or less extent. The
nucleus no longer appears serrated, but round. The nucleolus is now
much more sharply Outlined, and there is a considerable increase in the
amount of substance in the vicinity of the nucleus so that the cells appear
opaque. The glands themselves show similar changes. During a period
of rest its gland-cells are large and clear. Their nuclei are flattened and
parietal. The protoplasm is small in amount. The chief constituent
in the composition of the cells is a clear substance which represents the
secretion material of the gland-cells. When the gland becomes active the
nuclei become round. The nucleoli become more distinct, and at the same
time the nuclei are pressed more and more to the center of the cells. The
cells themselves become smaller on account of loss of the above-mentioned
clear substance. At the same time, there is an increase in the amount
of protoplasm, evidently the beginning of the production of a new secretion.
If we examine fresh material, instead of hardened preparations, we will

• We might mention in this connection the formation of retention cysts which often
take place in the parotid gland when the duct from a lobule becomes stopped up for
any reason ; for example, in case of inflammation. If the secretion of saliva were to be
regarded as a mere filtration process, it wovild be expected that the activity in the region
cut off would soon cease. The secretion, however, continues. The amount of pressure
developed in consequence is indicated by the swelling produced. Even if, later on,
secondary changes appear, creating new conditions, still for the beginning of the forma-
tion of cysts our observation holds true.

' Cf. A. Noll: Die Sekretion der Driisenzelle, Ergeb. Physiol, (.\sher and Spiro) Jg.
IV. p. 84 (1905).

' Zentr. med. Wissensch. 9, 130 (1866). Hermann's Handbuch der Physiol. 5, L
64 (1883).

488 LECTURE XXI.

obtain quite corresponding results. Thus in place of the above clear sub-
stance, little granules are noticed which represent the secretion material
produced by the cells. During rest these granules are being formed con-
stantly to be given off during activity. There has been a great deal of
discussion as to whether, in the secretion of the glands, th6 cells themselves
are destroyed, or whether it is to be assumed that the cell, as such, retains
its protoplasm and nucleus intact, and merely gives up the specific secre-
tion produced by it. According to all known observations, the latter
conception appears to be the correct one, for if the cells of the gland them-
selves should undergo breaking down, then there should be considerable
evidence in the active gland of a renewal of cell material. As a matter
of fact, there is but little sign of any such cell division taking place.

The cells of the various glands in the animal organism do not, to be sure,
show any marked difference from the cells of the other tissues. We
know that all sorts of different cells are constantly producing definite
products which take part in metabolism and in the exercise of particular
functions. We know, for example, that many cells give up ferments,
while others produce compounds of simple constitution; for example, the
cells of the suprarenal bodies produce adrenalin, and those of the intestine
form secretin. The formation of such products as an immediate con-
sequence of the activity of the cells escapes our observation only because
the amount formed is so small, and partly, as is the case with the
digestive ferments, because it is not these alone that are given up
by the cells, but there is a much larger amount of other material set free
simultaneously.

We may consider the formation of the secretion by the gland-cells in the
same light, and trace it to the activity of the cells, and in a narrower sense
to the protoplasm and cell-nuclei. We do not wish to speak — in order
to avoid misconceptions — of a transformation of protoplasm into secretion,
as is often done, but rather of the production of the latter by cell activity.
We must remember that the gland-cells are constantly being supplied with
new material by the blood, from which the secretion can be formed. This
is taken up according as the cell requires such material, and by means of
complicated processes the cell manufactures the secretion from it. The
protoplasm itself remains behind in the cell together with the nucleus;
both are preserved for a new formation of secretion. If we were to
assume that the cells themselves were passing over into the secretion, it
would be much more difficult to explain the process. There is no doubt
that ferments play an important part in the formation of this secretion.
Thus the cells of the gland have to break down to some extent the protein
substance in the serum in order to construct the mucin which is contained
in the secretion. The fact that evidently quite extensive transformations
take place is shown by the fact that mucin contains a large amount of

THE FUNCTIONS OF THE DIGESTIVE ORGANS. 489

glucosamine, while in the protein bodies present in serum there is but a
small amount of this aminohexose. It is conceivable that the albuminous
substances in blood-serum contain perhaps unknown preliminary stages
in the glucosamine formation, but it is, however, also possible that we have
here one stage in the process of the conversion of an amino acid into a
sugar. At all events, the formation of mucin with its peculiar composition
deserves considerable attention.

It is not to be assumed that during the activity of the gland, the cells
must be entirely built up as well as the secretion formed. At the same
time we should not think for a moment that these cells are permanent
structures. We do not doubt that they are constantly being renewed like
all other cells of the body, and that here and there a cell disappears to
be replaced by a new one.

The decrease in the volume of the individual cells during their activity
also has an effect upon the weight of the gland. If the submaxillary
gland is brought into activity by stimulating the fibers of the facial nerve,
it is found that the active gland decreases in weight.

Some attention is due to the question as to the manner in which inner-
vation of the salivary glands is normally produced. The Russian physi-
ologist Pawlow deserves great credit not only for having developed the
operative technique so that it is possible under purely physiological con-
ditions to trace the functions of the various digestive glands, but also for
having shown in an entirely original way how the activity of the same is
dependent upon definite external conditions. We have known for a long
time that the activity of the salivary glands is influenced by sensations
of taste and smell, and even by certain imaginations. Pawlow, however,
deserves the credit for clearly demonstrating by experiment the remark-
able ability that the salivary glands have for adapting themselves to their
work. Among other things he called attention to the following observa-
tions: If a dog is fed with dry, solid nourishment, there at once takes place
a considerable flow of saliva; while on the other hand, if the nourishment
is liquid, there results but a slight flow. Chemicals which act as irritants,
such as acids and alkalies, increase the flow of saliva in proportion to the
irritation produced. The organism attempts in this way to protect itself
from the action of such substances. It dilutes them and washes them out
from the mouth as much as possible. If small quartz pebbles are placed
in the mouth of a dog, the animal permits them to drop out gradually
from his mouth, but without any flow of saliva. If, on the other hand, the
same material is placed in the dog's mouth in the form of a powder, there
is a considerable flow of saliva. The purpose of this is clear, — in this way
the sand is washed out of the mouth, while in the case of the little stones
the tongue alone can accomplish their removal. In all such cases we are
struck with the utilitv of the whole mechanism. This is still more marked

490 LECTURE XXI.

when we find not only that the amount of saliva is regulated according to
the requirements, but likewise its composition. At one time it contains
considerable mucin, at another time relatively little, according to the con-
ditions. Apparently the irritation, whether it be chemical, thermal, or
mechanical, stimulates the apparatus at the end of the afferent nerves
in the mucous membrane of the mouth. From here the impulses are
transmitted to the central organ of the nervous system, and now by
means of the efferent nerves the individual salivary glands are called
into action. We speak of such a process in general as a reflex action.

It is of chief interest to us that it is possible by various outward effects
to influence the activity of the glands in such a way that the composition
of the saliva secreted is adjusted to the prevailing conditions. At
present we can only imagine how this adjustment may be effected.
The saliva itself always contains besides inorganic salts and water a certain
amount of organic material, as we have seen. We also repeat that
the saliva contains a ferment capable of hydrolyzing starch, the diastase.
It is remarkable too that it always contains small amounts of alkali
thiocyanate. We are wholly in the dark concerning the formation of this
last compound, or as regards its use.

In this connection it seems fitting to mention some observations con-
cerning secretions in the mouths of certain invertebrates. They are of
particular value because they make it easier for us to understand the most
important work of the gland-cells. Here we meet with the preparation
of strong acids by means of cell activity from material which could not
have contained the acid already formed. Long ago Troschel ' noted, in
examining a kind of snail, Doliuni galea, that the animal squirted
from its mouth a stream of liquid clear as water. The liquid showed a
strongly acid reaction, and caused effervescence on coming in contact with
the limestone lying on the ground. The secretion was produced from two
large gland-like organs lying near the stomach. The ducts from it ascend
on each side of the gullet and empty into the mouth. The secretion con-
tains sulphuric acid, and, in fact, as much as 4.1 per cent of the free acid is
present. Besides this, there is 0.4 to 0.6 per cent of hydrochloric acid.
Other varieties of snails similarly produce acids. Some of these " acid
snails " have been quite recently studied by Fr. N. Schulz.^ He followed
particularly closely the acid production on the part of the naked snail,
Pleurobranchia Meckelvi of the order Opisthobranchi. On being touched,
the snail rolls itself up. If the animal is squeezed a little, its external
surface after a short time becomes covered with a slimy secretion of acid
reaction. This comes from glands which are very numerous in the skin.

' Poggendorf's Ann. 93, 6U (1854) ; J. pr. Chem. 63, 170 (1854) ; see also de Luca and
Panceri, Compt. rend. 65, 577 and 712 (1867).
= Z. allgem. Physiol. 5, 206 (1905).

THE FUNCTIONS OF THE DIGESTIVE ORGANS. 491

Besides this secretion the animal empties from its pharynx a very strongly-
acid-reacting juice. This comes from a gland-like structure consisting of
long coils enveloped in a highly complicated, contractile network. The
chief constituent of the individual cells is a liquid, while the amount of pro-
toplasm itself is relatively small. If the gland is stimulated, the above-
mentioned network contracts; and the liquid secretion of the gland-cells,
which is contained in larger or smaller vacuoles, is emptied into the
exit duct. Evidently in this case the secretion is merely mechanically
removed from the cells. After the relaxation of the contracted cell-coil,
there remain in the cells at first only the shrunken protoplasm and the
cell-nuclei, and then begins anew the formation of secretion. Several
things indicate that the nucleus itself plays an important part in this
process. The secretion contains free sulphuric acid. What is the source
of this acid? It might come from sulphates, or from organic compounds
containing sulphur, such as, for example, albumin. The latter is hardly
to be considered as a possible source of sulphuric acid. It seems certain
that most of the acid must be formed from sulphates, for the amount of
acid produced is too large, and the amount of sulphur present in albumin
too small, to account for the formation by the assumption of an oxidizing
decomposition, as, for example, of cystine. It has been found that the
secretion continues during starvation. If the albumin itself were the
source of the sulphur, we would expect that the formation of sulphuric acid
would soon cease in the starved organism. It has never been explained
how the cells in the gland are able to produce the free acid. When we come
to discuss the formation of hydrochloric acid in the human stomach, we
shall find likewise that we are again in the dark. It has been attempted
to explain the formation of the strong acid as a result of the mass-action
law. We know that from salts of the mineral acids a small amount of
acid may be set free by the action of large amounts of carbonic acid, for
example. We also know that as a result of ionization a small amount of
acid ions are probabl)' set free in the organism. We shall not deny that
perhaps a part of the acid in the secretion may be formed in some such
way, and it is indeed conceivable that eventually all of the acid may be
produced in such manner, if we assume that as soon as a little acid is set
free, it is in some manner carried out of the range of chemical reaction, so
that constantly more and more of the acid will be formed. But even such
an hypothesis, which necessitates the further assumption of some means
of removing the acid as fast as it may be formed, does not enable us to
explain satisfactorily the whole process. At all events, the cells themselves
must exert a specific action. In this case, the cells of the gland form sul-
phuric acid alone, while in the stomach only hydrochloric acid results. It
might be assumed that the membrane of thecells is onlypermeable to certain
ions. It has. for example, been asserted that the walls of the stomach are

492 LECTURE XXI.

impermeable to chlorine ions, and in this way the formation of hydrochloric
acid in the stomach was explained.' It was soon apparent, however, that
such a theory was untenable. If we stop considering the production of
the acid by itself, but remember that the formation of the remaining
products of secretion point to a specific and extensive activity on the
part of the gland-cells, then evidently the formation of the free acid repre-
sents only one link in the chain of the entire secretion process. It is not
any more remarkable than, for example, the formation of mucin in the
cells of the salivary glands. Just as little as we are unable to account for
the formation of the latter as a result of purely physical or chemical
processes, is it possible for us to understand clearly the production of acid
on the part of the cells.

The question next arises concerning the biological significance of the
acid secretion in snails. Troschel, the discoverer of the presence of the
acid, was inclined to believe that it was a means of protection against
enemies. This is hardly correct in the sense meant. Although the
Doliiim galea is able to throw out this secretion when on land, the irritating
effect of the acid would be lost when the animal is in the water, and,
moreover, the water itself offers so much resistance that obviously the
animal would not be able to .send out a stream of secretion at any desired
moment. On the other hand, it is possible that the acid formation, espe-
cially on the part of the glands in the skin of the Ple.urobranchi, may serve
indirectly as a means of protection in the sense that by reason of it these
snails will be avoided as food by other animals. Unquestionably, the
secretion of the large glands in the gullet must have some other significance.
It has been suggested that they play a role in digestion- Careful investi-
gation, however, has proved that this is not the case. Semon^ believed
that the sulphuric acid acts upon the lime skeletons of other animals upon
which these snails feed. Thus calcium sulphate would be produced.
The effect of this decomposition Semon studied on the skeleton of the
star-fish. He found that the latter, after it had lain for some time in
water containing sulphuric acid, could be broken up by means of the
fingers. The direct examination of the intestinal contents of these snails
did not lend any support, however, to the assumption of any such action.
It is, therefore, improbable that the sulphuric acid is produced for the
purpose suggested by Semon. Now we find in the animal kingdom
repeated examples of contrivances by means of which one animal which
eats another .species for its nourishment is able to cripple its prey. This
is the case with the poison glands of snakes. Perhaps in the case of the
snails tiie acid forms a weapon of attack.' Many sea animals are extremely

' Cf. Koppe.Pfluger's Arch. 62, 567 (lS92),and Landislaus v. Rohrer: ibid. 110, 416 (1905).

= Biol. Zentr. 9, 80 (1890).

" W. Preyer: Naturwissensch. Wochschr. Berlin 5, 481 (1890).

THE FUNCTIONS OF THE DIGESTIVE ORGANS. 493

sensitive to acid. Thus tlie Echinoderms draw in their suckers when
exposed to acid. They are then easily removed from the place to which
they had attached themselves.

Let us now return to the saliva. We have already mentioned its most
important functions. They are chiefly of a mechanical nature, — the
food is surrounded by saliva and thus made easy to swallow. The saliva
also is an important means of keeping the teeth clean. In this case also
it exerts first of all a mechanical action, and, on the other hand, when of
normal composition, it also tends to prevent decomposition by the bacterial
flora of the mouth. It is probable that in many cases the formation of
caries in teeth is due to an abnormal composition of the saliva. Tooth
decay, moreover, usually results from a faulty formation of the tooth itself.
The tissue composing the teeth is closely related to that of the bones.
Three tissues are known to take part in the formation of teeth, of which one,
the cement, corresponds to the bony tissue. Dentine also has a similar
composition. We find that bones contain: calcium phosphate, magnesium
phosphate, calcium fluoride, calcium chloride, calcium carbonate, ferric oxide,
and an organic basal substance, which on boiling yields gelatin. Enamel,
the third tissue of teeth, is characterized by its containing the least water
and the most mineral matter of any substance in the human body. Under
normal conditions it exerts great resistance to external influences, and, as
long as it is intact, prevents infection by bacteria.' The enamel contains
lime salts chiefly. At this place we need hardly mention the importance
of the food being well chewed. \t is perfectly clear that the subdivision
of the food is of great benefit as regards the rapidity with which it is acted
upon by the digestive ferments. By means of the teeth the food is changed
into such a state that it can be acted upon readil}^ by the digestive juices.
It might be thought that on feeding with broth, the function of the teeth
would be replaced. We shall see later on, however, that the manner in
which the food is prepared has a great influence upon the function
of the secretions in the digestive tract, especially that of the stomach.
A uniform diet could not possibly stimulate our senses permanently.

In many cases the saliva acts as a solvent, and in this way the sensation
of taste is obtained, for we can only taste substances which are in solution.
The peripheral organs of the sense of taste are distributed throughout the
whole of the mouth. We find them on the upper surface of the tongue,
on the under surface at the tip of the tongue, in the mucous membrane of

' We should not fail to mention in" this case the remarkable readiness with which
wounds in the mucous membrane of the mouth, or indeed of the entire alimentary
canal, are healed ; and how seldom there is any infection here in spite of frequent ex-
posure. We may perhaps speak of the immunity of cells in the mucous membrane
and surrounding tissue. This immunity may be acquired by the fact that metabolic
products of bacteria in the mouth are absorbed from the dilute solution there, and this
creates immunity.

494 LECTURE XXI.

the soft and hard palate, the anterior pillar, the tonsils, the posterior pillar, '
the uvula, the epiglottis, and even the throat, itself. This wide distribution
of the organs for taste perception is only noticeable during youth. In
adults the sensation is more localized, although it varies greatly with indi-
viduals. The mucous membrane of the cheeks, the uvula, tonsils, and
the middle of the tongue, are almost always incapable of taste perception
in adults. The end-apparatus of the taste-nerves form the so-called taste-
buds or taste-goblets. In man the glossopharyngeal and trigeminal nerves
have been recognized as taste-nerves. The former innervate the back
part of the tongue, the latter the front part. Individual peculiarities are
noticed here, and sometimes one of these nerves alone provides for the whole
region. The different sensations of taste may in general be attributed
to four different qualities; namely, sweet, sour, bitter, and salt. We also
speak of alkaline and metallic tastes. There is hardly room for doubt that
these different sensations of taste are produced by the aid of different
nerves, so that for the taste-nerves the laic oj specific sense energy ' applies.
According to this law, one and the same excitant when acting upon different
nerves will always produce different sensations, while, on the other hand,
all sorts of different excitants always produce the same sensation when
acting upon the same nerve. The different quality of the sensation is
therefore merely caused liy the different nature of the end-apparatus in
the central nervous system. Moreover, the peripheral reception appara-
tus is so specialized that it is only affected by certain definite excitants.
It might be thought, and especially in the study of the sensation of taste,
that the chemical constitution of a substance having a definite quality of
taste might give us some idea of the way in which the definite end-apparatus
is excited. Numerous experiments have been performed with this idea
in mind, but without success.^ Thus, for example, a great many amino
acids taste sweet, while others are bitter. Glycocoll, alanine, and a-amino-
valeric acid are sweet, while Z-leucine, which occurs in nature, is bitter.
It is remarkable that rf-leucine has a sweet taste.' In (^Meucine the sweet
taste predominates. The sense of smell is closely related to that of taste,
and frequently they are confused with one another. It is, furthermore,
interesting that both these sensations of taste and of smell may be pro-
duced by means of a very small amount of material. The organ of smell
is in some cases especially sensitive. Emil Fischer and Penzoldt found,
for example, that as little as 0.000,000,04 milligram of mercaptan in a

' Johannes Miiller: Zur vergleichenden Physiologie des Gesichtssinnes. Leipzig
(182()). Cf. R. Weinmann: Die Lehre von den specifischen Sinnesenergien, Hamburg
and Leipzig (1895).

' Cf. Wilhelm Sternberg: Arch. Anat. Phys. 1903, 5.38; 1904, 483; 1905, 201; Bar.
15, 36 (1905).

' Emil Fischer and Otto Warburg: Ber. 35, 3997, 4005 (1905).

THE FUNCTIONS OF THE DIGESTIVE ORGANS. 495

liter of air is perceptible. We shall repeatedly come back to the influence
of the sensatory impressions upon the functions of the digestive glands.
The nerves of smell and of taste are important protective organs. They
call attention to processes of decomposition, to decay in our food, and allow
us to recognize the presence of many injurious substances.

From the mouth, the food is carried by the process of swallowing into
the oesophagus, and from here directly into the stomach through the cardia.
With the exception of a slight transformation of starch into sugar, the
real process of digestion does not begin until the food reaches the stomach,
where it proceeds energetically. We repeat that the diastase from the
saliva can under certain conditions continue its action for some time, but,
on the other hand, the stomach itself has its own ferments. As has been
recently shown, it possesses, for one thing, a lipolytic ferment, although we
have at present no means of estimating the extent of its activity. Its sig-
nificance has been quite variously estimated and, in fact, its very presence
has been doubted by some. Besides lipase, there are ferments present in
the stomach which are capable of acting upon the protein of the foods.
These are pepsin and rennin. We have already stated that the existence
of the latter ferment has been quite i-ecentl}' questioned, and it has been
assumed that the two properties of coagulating milk and dissolving albu-
min are due to the action of a single ferment. We can adopt this assump-
tion that the action of rennin corresponds to that of pepsin, only when
it has been verified by further investigation. Until this has been done,
we will content ourselves with the older conception of the presence of both
pepsin and rennin, although, we have already seen, there are a number of
facts which speak in favor of Pawlow's hypothesis.

These ferments are produced iiy certain glands in the mucous membrane
of the stomach. The stomach itself is not a physiological unit. Even
the outer appearance shows a marked difference between the pyloric and
fundus (or cardiac) portions of the mucous membrane. That of the
former is pale and has a few deep folds, while the latter is of a reddish-
yellow or reddish-gray color, and has numerous folds which are connected
with one another by a sort of network. In these net-like little hollows
between the folds end the stomach glands. There are two types of these
glands, one of which contains but a single kind of cell, while the other
contains two. In the pyloric end there is found but one cell form, while
in the cardiac or fundus end the glands are of the latter type. There is
no sharp distinction between them, however. These two types of glands,
both of which are tubular in shape, are named according to the locality
in which they are found, and are known respectively as pyloric and cardiac
or fundus glands. The former contain a cylindrical epithelium, and the
latter contain, in addition, other smaller cells irregularly distributed
between the larger cells and the basement membrane. The first kind of

496 LECTURE XXI.

cells are designated in the cardiac glands as chief, central, principal, or
adelomorphous cells, while the latter are called either border, parietal,
or delomorphoiis cells. Between the gland-cells there are fine secretion
capillaries which surround the border cells like a basket. For a long
time it was believed that this histological distinction of two kinds of cells
also corresponded to a physiological distinction. The fundus glands were
supposed to secrete only pepsin, while the pyloric glands merelj' formed
mucous. That this is not the case is shown by the fact that the secretion
carefully collected from the latter portion of the stomach does actually
contain pepsin. The pyloric and cardiac parts can easily be isolated, and
in this way " small stomachs " are formed in the Pawlow sense, and by
means of fistulas the contents of each may be studied by itself. At
present we are still undecided as to the especial significance of the chief
cells and of the border cells. It has been established that the former
take part in the formation of pepsin and rennin, but the function of the
border cells remains vague. From the fact that the pyloric part of the
stomach produces little or no hydrochloric acid, it has been suggested that
the border cells produce the acid, but there has never been any direct
proof of this.

Whatever the functions of the cells may be, it is to be said that much
the same changes take place in them during activity as in the cells of
the salivary glands. During a period of rest the secretion is formed
which is given up during the period of work.

All the secretions of the mucous membrane of the stomach and its
glands taken together form the so-called gastric juice. It consists in part
of mucous which is given off chiefly from the superficial surface of the
mucous coat, together with the ferments, hydrochloric acid, inorganic
salts, and small amounts of other organic substances. Exact studies
concerning the formation of the gastric juice and of its dependence upon
outward influences were first made possible by the operative skill of
Pawlow and his pupils. His methods and his investigations gave us the
first insight into the relation between the secretions of the stomach
and physiological conditions. Pure gastric juice may be obtained by the
establishment of a fistula, best in combination with one in the cesophagus.
Thus the food swallowed by the animal may be removed before it reaches
the stomach. In this way a fictitious feeding is obtained, and the gastric
juice is not contaminated with the food, nor with the intestinal products.
It was found advisable also to isolate a portion of the stomach, forming
a little stomach, or blind sack, in which the secretion process could be
studied by means of a fistula while digestion was going on in the rest of
the organ. The gastric juice flowing out of such a fistula is, after filtering
off the mucous, clear as water, odorless, and of an acid taste. This taste
is due, as has been stated, to the presence of free hydrochloric acid. In

THE FUNCTIONS OF THE DIGESTIVE ORGANS. 497

dogs there is from 0.46 to 0.6 per cent acid present, while in man the
values vary greatly. Thus the amount present has been variously stated
in the literature as from 0.05 to 0.57 per cent. We have already said
that it now seems impossible to regard this free acid as taken directly
from the blood, for the blood may be considered as of neutral reaction.
A great number of experiments have been undertaken without definitely
settling this question of the acid formation. We must rest content with
the hypothesis that it is due to a specific activity on the part of the cells,
and will state once more that it does not imply a process any more com-
plicated than that of the formation of other substances which result from
the specific activity of the cells in every gland. There is no justifiable
ground for giving a peculiar position to the acid secretion.

The individual ferments are not found in a finished state in the mucous
coat of the stomach; i.e., they are not given up by it in an active condi-
tion. This preliminary condition of the ferment is spoken of in general
as that of a zymogen, and in the case of pepsin itself we have pepsinogen.
The presence of such a substance may be shown by the following experi-
ment: Pepsin itself is extremely sensitive to a solution of soda, by
means of which it is soon destroyed. Pepsinogen, on the other hand,
resists the action of soda much more strong!}'. If the mucous membrane
of the stomach is cut up into small pieces and extracted with a dilute
soda solution, there is obtained after filtering a liquid which of itself exerts
no digestive action upon albumin, but does so on being acidified with
hydrochloric acid. The acid serves to convert pepsinogen into pepsin.
If, now, after the digestion has proceeded for a short time, the liquid is
again made alkaline, a further addition of acid will no longer bring forth
the digestive action of pepsin. The pepsin has been destroyed by the
soda. We are at present unable to explain how the acid activates the
zymogen, and, in fact, such processes will remain obscure until we know
more about the chemical nature of the ferments themselves. We can
indeed assume that the hydrochloric acid in some way causes a rearrange-
ment of the atoms in the ferment molecule, possibly by the formation of
an anhydride or something similar, and that in the new state the ferment
is capable of exerting its characteristic action.

Rennin likewise is not present in the mucous membrane as such, but in
the form of a zymogen. It is activated in precisely the same manner as
pepsinogen, and in fact these two ferments are very similar in their entire
behavior.

It is highly significant that the function of the mucous membrane and
its glands is dependent upon definite kinds of stimulation. These can be ef-
fected in part in the stomach itself, or the stimuli may be transmitted to the
stomach from other organs. The secretion of the gastric juice is brought
about by reflex action. This may be demonstrated very prettily by making

498 LECTURE XXI.

oesophageal and gastric fistulas in a dog. Before the animal is fed, no
gastric juice flows out of the latter. When the animal is offered food,
first of all the dog chews it; on swallowing, instead of reaching the stomach,
the food passes out through the fistula in the oesophagus. Thus there
is no chemical, thermal, or mechanical stimulation produced upon the
mucous membrane of the stomach. Notwithstanding, five or six minutes
after the animal is offered food, there takes place regularly an abundant
secretion of gastric juice. Pawlow and Schumow-Simanowskaja ' have
also succeeded in proving that the vagus nerve carnes secretory fibers.
The production of gastric juice is not entirely dependent upon the vagus
as is evident from the fact that it continues after both the vagi have been
severed, although the quality of the secretion is apparently changed; at
least, there is less pepsin in it.

Highly important is the observation that psychic influences have the
decisive effect upon the formation of gastric juice. It may be shown that
the action of the nerves of taste and of smell alone do not suffice to cause
a secretion from the lining of the stomach. Similarly the act of chewing
is ineffective of itself. The reflex secretion of the stomach juices is only
effected when the animal has the desire to eat. If, after the latent
period of about five minutes, the secretion of gastric juice has once begun,
it then continues for two or three hours. This secretion produced reflex-
ively ceases immediately on cutting the two vagi nerves. It would be
wrong to assert that the nerves of smell and of taste take part in the psy-
chical stimulation of the secretion. To be sure, they may aid indirectly
in many cases, by recalling to the imagination certain impressions which
awaken the desires to eat. Of course these impressions may in some cases
be first caused by the momentary stimulation of smell or taste.

By means of such experiments as those carried out by Pawlow in many
directions, the great importance of the appetite has been clearly estab-
lished. It is by no means a matter of indifference whether one eats with
pleasure or from compulsion.

The importance of the gastric secretion produced reflexively by psy-
chical influences is made very clear by the following experiment performed
by Pawlow. He took two dogs, each having fistulas in the oesophagus and
in the stomach, and introduced equal-sized pieces of meat through the
gastric fistula of each dog in such a way that the animal was not conscious
of the operation. One of the dogs was then given a piece of meat to devour.
(In such cases, where the food devoured does not reach the stomach of the
animal, we speak of a fictitious meal.) After some time had passed, Pawlow
compared the pieces of meat in the stomachs of the two dogs. It was
found that the meat in the stomach of the dog which had received the

Arch. Anat. Physiol. 1895, 53.

THE FUNCTIONS OF THE DIGESTIVE ORGANS. 499

fictitious meal was much more thorouglily digested tlian the meat in the
stomach of the other dog.^

It is an old experience that psychic influences may destroy the appetite.
In this case there are great individual differences; often slight anger will
suffice to destroy the appetite completely. These experiences can be shown
experimentally to be well founded. Thus, among others, A. Bickel ^ has
found that the gastric secretion stops at once when a dog is confronted
with a cat. Undoubtedly the same relations exist in man. Naturally,
in the latter case we do not possess such a rich field for observation. In
man such experiments are influenced much more by secondary effects than
is the case with animals, where reactions take place more in accordance with
sensatorj' impressions. Certain imaginary effects are not so prominent
in animals as in man. On the other hand, such experiments carried out
with human beings are less valuable, because, when there is a gastric fistula,
there is usually some pathological derangement of the stomach or (Esopha-
gus. Frequently there are tumors, especially cancers, to be considered.
The latter are especially likely to cause a most deep-seated effect upon the
whole metabolism of the cells and weaken the whole body, so that it is out
of the question to speak of a normal function of the lining of the stomach
and of its glands, even although the carcinoma may not have attacked
the stomach itself. On the other hand, now and then the formation of a
gastric fistula becomes necessary when there is no chance for the food to
reach the stomach through the cesophagus, for example, as a result of
strictures caused by injury to the mucous membrane. In such cases it is
possible to carry out observations with human beings which are similar to
those of Pawlow with dogs. Thus, Hornborg,' who studied a case of gas-
tric fistula with oesophageal stricture, observed that it was not possible to
detect psychic influences in all cases. The chewing of substances with
pleasant taste produced a secretion, while chewing of indifferent or badly
tasting substances had no effect. Chewing of itself appeared to act favor-
ably upon the gastric secretion, while the mere sight of food had no effect.
On the other hand, the secretion stopped if the boy was not allowed to eat
at once something that tasted good; this evidently made the boy angry,
and this feeling was indicated by a flow of tears.

The secretion of the gastric juice is produced not merely as a result of a
reflex action, but we recognize certain other influences as well, which may
be exerted within the stomach itself. Mechanical irritation does not
suffice to start the flow of the juice. Chemical influences alone are to be

' The highly interesting studies hy Pawlow and his students have nearly all appeared
in Russian only. His lectures have been translated into German by A. Walther, and
published in 1898 by Bergmann of Wiesbaden.

» Deut. Med. Wochschr. 31, 1829 (1905).

3 Inaug. Dissert. Helsingfors (1903).

500 LECTURE XXI.

considered in this connection. Digestive activity is especially favored
by broths, extracts, juice of meats and milk, and is somewhat stimulated
by water and small amounts of alcohol. Fats, on the other hand, restrain
the production of the digestive fluid, and also influence its qualitative and
quantitative composition. These effects may be observed to best advan-
tage by introducing the different substances into the stomach of an
animal without its knowledge. The secretion under such conditions does
not take place as quickly, usually not until after 15 to 30 minutes,
and persists for various lengths of time according to the nature of the
food. Experiments of this nature give one the impre.ssion that such
a direct stimulation of the glands is incomplete in effect. A har-
monious course of digestion is only assured when the secretion is strongly
stimulated reflexively. We can indeed imagine that by means of
digestion itself, substances are formed constantly which act as chemical
irritants upon the lining of the stomach and the glands, so that after
the flow of the juice is once started, it continues for a considerable
length of time.

Pawlow and his students obtained very interesting results when they
attempted to ascertain the effect of different kinds of food. It was found
that the cells of the stomach did not produce the same juice in all cases.
On the contrary, the composition of the digestive juice was suited to the
nature of the food. First of all, it is of interest to know that the acidity
of the juice remains constant in the secretion, while the amount of ferment
present varies greatly. Now it is a well-known clinical fact that widely
divergent degrees of acidity are found in the contents of the stomach.
Such determinations, however, are of but a slight value, for many reasons;
on no account should they be used to indicate the amount of hydrochloric
acid normally present in the gastric juice. As we have already men-
tioned, it is not right to regard the contents of the stomach as a uniform
mixture of the digestive secretion and the food. As Griitzner ^ has recently
proved, the newly-introduced food stands in the midst of that which has
been in the stomach for some time, and does not at once come in contact
with the walls of the stomach. In the pars-splenica of the stomach, the
food may remain for hours without coming into intimate contact with the
gastric juice. If now a part of such a digesting mixture be siphoned out
of the stomach, it is obvious that a determination of the degree of its acidity
might easily give rise to false conclusions. Quite a number of factors
here come into play which may easily conceal the fact of the original
uniformity in the acid content of the juices. It has been observed,
for example, that with dogs in which there was a vigorous secretion, there
was more acid in the gastric juice than when the production of the same
took place more slowly. Similarly a higher acidity was noted if the juice

' Pfliiger's Arch. 106, 403 (1905).

THE FUNCTIONS OF THE DIGESTIVE ORGANS. 501

was taken directly from the fistula than when the latter was closed for a
time. The reason for this can be explained. The gastric juice flows over
the walls of the stomach, which are covei-ed with alkaline mucus, before
■ it. reaches the fistula. When there is a considerable amount of the juice
being secreted, it is obvious that proportionately less hydrochloric acid
will be neutralized than when only a small amount of juice passes over
these walls; and similarly when the fistula is closed, the acid is more com-
pletely neutralized by the alkaline mucus than when it passes off freely.
As Pawlow has stated, in a normal stomach as much as 25 per cent of the
original acidity may be neutralized by the mucus. There are, to some
extent, very complicated processes concerned in this neutralization the
significance of which cannot be entirely disregarded. It is indeed possible
that certain relations exist between the hydrochloric acid content of the
stomach juices and the formation of the mucus by the membrane of the
stomach, and that here again there is an adjustment corresponding to
the nature of the different foodstuffs. Naturally the deviations in the
acidity of the stomach juices var}' much more greatly after the food has
reached the stomach. At all events, any values obtained in this case
should be very cautiously applied to the composition of the juice itself.
The clinical practitioner must always have in mind all sorts of different
relations, and should determine the combined hydrochloric acid as well
as that which is still free. The careful physician should never be satisfied
with a single observation, but should base his judgment upon examina-
tions carried out under the most varied conditions.

Pawlow calls attention to the adaptability of the whole work of the
stomach, and especially of its glands. This is shown in a number of little
ways, and we are able to trace the functions of the stomach very well
because we know the condition of the food as it enters. Such relations
are much more difficult to establish in the study of the pancreas, and in
some cases it is impossible, because its juices come in contact, under
normal conditions, with an inextricable mixture consisting partly of
decomposition products, and partly of unchanged food. Investigations
have shown that a mixed diet, as well as the feeding of single articles, such
as milk, bread, meat, etc., leads to a perfectly definite formation
of the gastric juice. This is true not only of the composition of the fluid,
but of the amount secreted and the duration of the secretion. First of
all it is to be noted that the amount of gastric juice secreted is practically
proportional to the amount of food. Thus 100 grams of raw meat caused
the secretion of 26.0 cubic centimeters of juice; 200 grams = 40.0 cubic
centimeters; 400 grams = 106.0 cubic centimeters. For a mixed diet,
composed of milk, bread and meat, the following values were obtained: —
100 cubic centimeters of milk, 50 grams meat, and 50 grams of bread,
correspond to 42.0 cubic centimeters of gastric juice, while double the

502

LECTURE XXI.

above quantity of the mixture caused the secretion of 83.2 cubic centi-
meters.

The digestive power of the gastric juice depends greatly upon the nature
of the food. Pawlow and his students studied the power of digesting
albumin on the part of both the gastric and pancreatic juices by Mett's
method. This consists of sucking up the white of an egg into glass tubes
of 1 to 2 millimeters bore and then coagulating it at a definite temperature.
These tubes are inserted under entirely corresponding conditions into the
digestive liquids, and taken out at the end of a definite period. Then,
by means of a millimeter rule and a microscope, the amount of albumin
that has become digested is measured. The following values are given
as examples of such an experiment:'

At 8 o'clock the animal experimented upon was fed 200 grams of bread.
It secreted the following amounts of gastric juice with varying digestive
power:

Time.

Amount of Juice per Hour.

Digestive Power.

8-9 o'clock

9-10 o'clock

10-11 o'clock

3.2 c.c.
4.5 c.c.
1.8 c.c.

8.0 ram.
7.0 mm.
7.0 mm.

The same dog was then fed 200 grams of raw meat:

Time.

Amount of Juice per Hour.

Digestive Power.

12 o'clock

1 o'clock

2 o'clock

8.0 C.c.
8.8 c.c.
8.6 c.c.

5.37 mm.
3,50 mm.
3.75 mm.

Then 200 cul)ic centimeters of milk were fed to it:

Time.

Amount of Juice per Hour.

Digestive Power.

3 o'clock .

4 o'clock .

9.2 c.c.
8.4 c.c.

3.75 mm.
3.30 mm.

A control experiment showed that the values given were in no way
caused by the order in which the food was eaten. It is evident from these
figures that the juice secreted after feeding with bread possesses the greatest
digestive power. Milk produces the weakest secretion of all.

' Cf. J. P. Pawlow: Die Arbeit der Verdaungsdriisen, p. 42. P. Chigin: Arch, des

sciences bid. Ill and Inaug. Diss. St. Petersburg (1894).

THE FUNCTIONS OF THE DIGESTIVE ORGANS.

503

The total acidity likewise varies according to the nature of the food.
It is greatest with meat and least with bread. It is interesting also to
note that the duration of the secretion is also regulated according to the
nature of the food; and in fact during the course of secretion, from hour
to hour, the composition adjusts itself qualitatively to the conditions.
We will give here the result of another of Pawlow's experiments : '

AMOUNT AND NATURE OF GASTRIC JUICE WITH DIFFERENT
NOURISHMENT.

200 gms

Meat, 200 gms. Bread, 600 c.c. Milk.

Hours.

Amount of Fluid in c.c.

Digestive Power in

mm.

Meat.

Bread.

Milk.

Meat.

Bread.

Milk.

1

11.2

10.6

4.0

4.94

6.10

4.21

2

11.3

5.4

8.6

3.03

7.97

2.35

3

7.6

4.0

9.2

3 01

7.15

2.35

4

5.1

3.4

7.7

2.87

6.19

2.65

5

2.8

3.3

4.0

3.20

5.29

4.63

6

2.2

2.2

0.5

3.58

5.72

6.12

• 7

1.2

2.6

3.25

5.48

8

0.6

2.2

3.87

5.50

9

0.9

5.75

dO

0.4

From these figures it is evident that the maximum secretion is produced
b}' meat during the first or second hour. In the case of bread, the max-
imum secretion is reached during the first hour, while with milk it is the
second or third hour. With meat the juice secreted during the first hour
has the greatest digestive power, in bread it is that of the second and
third hours, while with milk the maximum digestive power is obtained
much later.

These values do not merely correspond to a single experiment. They
have been obtained again and again. At present we cannot say anything
concerning the significance of these variations. We can indeed assume
that one food requires more ferment for its hydrolysis than another, in
order that its decomposition may take place to an equal extent within a
given period. We must admit, however, that we are here confronted
with many problems which cannot be solved until we know more concern-
ing the nature of the ferments themselves and of the fermentation processes
which they cause to take place. We introduce these interesting observa-
tions here, only to show how well organized are the functions of the diges-
tive glands. In this way it is easier for us to understand, in considering

' Loc. dt. p. 44.

504 LECTURE XXI.

physiological processes, how great an effect is produced by any disturbance
in the functions of the stomach. It is now clear to us that severe gastric
disturbances may be brought about by purely nervous influences without
there being organic changes. It is easy for us to believe that a hyper-
secretion may be produced by a condition of stimulation, caused, for
example, by a supersensitiveness of the secretory fibers of the vagus. On
the other hand, the experience of Pawlow and his school indicates the possi-
bilities of conditions of restraint with a limited supply of secretion. The
fact that the cells of the stomach glands are extremely sensitive to chem-
ical stimulation, and adjust themselves to the nature of the nourishment
with regard to their entire activity, enables us to understand that under
pathological conditions it is not necessary for the amount of secretion to
have become decreased or increased. Disturbances may arise which affect
the production of some particular substance. It is perfectly clear that
under such conditions the entire adjustment of the secretion to the nour-
ishment would be affected. Thus, normally, for the digestion of bread,
but little hydrochloric acid is present in the stomach during' the entire
duration of the secretion. There is, to be sure, a reason for this, for by this
means the digestion of starch by the diastase in the saliva will continue
much longer.

It is absolutely necessary that we should give prominence to the researches
of Pawlow in our study of digestion in the stomach. By means of
them, all the observations which have served for a long time to establish
the existence of characteristic sense-nerves, may be applied likewise to the
innervation of the intestinal canal and its glands. These organs also
do not react fully by means of a single stimulation. Here again the stim-
ulation is only taken up by certain definite cells and transmitted in a
perfectly definite manner. The I'esults of Pawlow's experiments are not
at all astonishing. We may assume that purely chemical processes play
a prominent part here. We may imagine that a certain kind of cell is
adjusted so that it is susceptible to a given chemical stimulation, while
a different cell is affected by another chemical substance. We may per-
haps apply the facts that we have established in the study of ferments
directly to the cells as a whole. The ferments are likewise products of
the cells. The individual cells produce them in such a way that they
possess certain groups which can react only with definite compounds
corresponding to a characteristic grouping. Conversely, the cells may be
so constructed that their function as a whole only appears when started
by the action of certain definite substance.

The more extensive our knowledge becomes, and the more we enter
into the secrets of the metabolism of cells, the better we become con-
vinced that the cells themselves act by means of ferments. They do not
part with such ferments, but retain them for their own use. These cell-

THE FUNCTIONS OF THE DIGESTIVE ORGANS. 505

ferments may perhaps have a zymogen state of existence which requires
an activator to bring its action into play. Every cell may possess several
ferments. One substance may serve to activate a given ferment, while
another activates a different one. Again, the gland-cells act by the aid
of ferments. Thej' are broken down and again built up until from the
building material, which is of quite different composition, the specific
secretion is produced. Now the cells of the glands in the stomach produce
the secretion during a period of rest. Thej' retain it until, by means of
some sort of stimulation, they are made to give it up. We must not
imagine that the act of secretion itself is a purely physical process. The
material of which the secretion is composed does not exist in the cells in
a condition capable of exerting the characteristic function. Probably
diu'ing the secretion activity, a group of atoms is eliminated here and
there and new combinations are effected. We do not yet know whether
all these cells of a gland contain the same secretion, or not. All such
assumptions are but speculations, without any experimental foundation.
We mention them merely because the first glance at the results of Paw-
low's investigations, which almost lead one to assume that the digestive
glands are furnished with intelligence, must give one the impression that
we are meeting with conditions here which are infinitely complicated, and
which will be, apparently, inaccessible to further investigation. This is
not really true. We have no doubt that from these experiments of Paw-
low the first light will be shed upon the great obscurity which enshrouds
the functions of the glands and their dependence upon nervous influences.
To be sure, we are still far from the goal. Pawlow deserves our thanks
for having at least pointed out to us the ^i'ay this is to be attained.

We have up to now been concerned chiefl}^ with pepsin, which is brought
into activity by acid, and which is extremely sensitive to alkali. Now it
is known that the pylorus part of the stomach secretes no acid. In spite
of this fact, there is digestive power in the juice produced at this region
of the stomach, as has been shown by experiments with an isolated blind
sack in the pylorus. It is of much interest to find by the experiments of
Pawlow and Parastschuk '■ that the proteolytic ferment of the pylorus
digestive juice also requires hydrochloric acid to activate it. The acti-
vated juice shows a proteolytic and milk-coagulating action. It has been
asserted that the pylorus portion of the stomach secretes a ferment which
is active in alkaline solution.- If this be true, then we shall beiorced to
assume that a ferment other than pepsin is produced in this portion of the
stomach. At present, however, we do not have suflficient ground for believ-
ing that there is actually a different ferment here, for it seems far more
probable that the mucous membrane of the pyloric region, or the glands

' Z. physiol. Chem. 42, 415 (1904).

' Karl Glassner: Hofmeister's Beitr. 1, 24 (1904).

506 LECTURE XXI.

there, secrete pepsinogen, which comes into action only when brought into
contact with the acid juices of the stomach. We do not yet have the means
at our command for arranging the proteolytic ferments into classes with
satisfactory exactness. We can, however, distinguish between ferments of
the pepsin class and those similar to trypsin. The best way of establish-
ing the class to which a ferment belongs, is, in this case, to allow it to act
upon a polypeptide, and the results from the experiment are obtained most
readily if we choose a polypeptide in the formation of which a difficultly
soluble amino acid, e.g. tyrosine or cystine, takes part. GlycyW-tyrosine
is decomposed in a short time by trypsin and similar ferments, but this
dipeptide is not acted upon by pepsin. The secretion of the pyloric region
behaves quite like the latter after it has been activated by hydrochloric
acid.^ It seems certain, therefore, that the ferment of the pylorus cells
belongs to the pepsin group, as was assumed by Pawlow.

Now that we have become acquainted with the influence of the food,
and its nature, upon the secretory relations in the stomach, it is time
for us to turn to the action of the gastric juice upon the food itself. We
have discussed this already at some length in considering the different
classes of foodstuffs. Here we will merely repeat that pepsin, with the
aid of hydrochloric acid, converts the albumins chiefly into peptones and
in part to simpler cleavage-products; but on the other hand, a breaking
down of the simplest cleavage-products, i.e. amino acids, cannot take
place here, or at least only to a very slight extent. Again, the lipase causes
the hydrolysis of a part of the fat, and in this way prevents, to some
extent, the restraining influence which the fats have upon the gastric
secretions. The carliohydrates, furthermore, may be decomposed some-
what while they remain in the stomach, but, to be sure, not by means
of the ferment obtained from the gastric glands, but rather by means of
the diastase from the saliva. This diastase, however, is destroyed on
coming in contact with the acid of the stomach. Its period of action,
therefore, depends upon the acidity of the gastric juice and the nature of the
food. In case the food is in the form of a loose mixture which is easily mois-
tened, it is evident that the action of the diastase cannot long continue.

Under the influence of the gastric juice, the food is changed into a pulpy
mass known as the chyme. This consists, in part, of products formed
from the decomposition of the food, and in part of unchanged food. For-
merly it was thought that the muscular activity of the stomach had a
great deal to do with the formation of this chyme. Doubtless in this way
the contents of the stomach are thoroughly mixed and brought into inti-
mate contact, layer by layer, with the gastric juice, but, on the other
hand, this process takes place gradually, and not by means of violent
muscular contractions, so that it is not right to speak of the food being

E. Abderhalden and P. Rona: Z. pliysiol. Chem. 47, 359 (1906).

THE FUNCTIONS OF THE DIGESTIVE ORGANS. 507

kneaded in the true sense of the word. The innervation of the musculature
of the stomach is partly provided by the vagus and partly by the sym-
patheticus. Since even the extirpated stomach contracts spontaneously,
it has been assumed that the ganglion-cells in the walls of the stomach can
cause this action.'

After the chyme is formed, the stomach has fulfilled its task. The
pj''lorus then opens and the chyme enters the duodenum. This trans-
ference does not take place all at once. The time that the food remains
in the stomach depends upon a number of factors. Purely physical
conditions, such as the size of the food particles and the chemical nature
of the contents of the stomach, both have an effect.^

Frequently we hear of a foodstuff being easihj digestible or difficultly
so without its being perfectly clear just what is meant by the term.
As a matter of fact, it depends upon two factors. A food may be readily
digestible, i.e., it can be readily acted upon by the ferments in the stomach,
and yet appeal to us, according to its entire behavior, as difficultly diges-
tible. This is due to the fact that although it may be easy for the ferments
to act upon a food, still it may be converted into chyme only with consider-
able difficulty. The readiness with which a food may be converted into
chyme should always be considered with regard to its digestibility. Diges-
tion experiments in a test-tube cannot decide this. Many contradictions
in theory and practice are to be traced to this point. Our present knowl-
edge concerning the digestibility of various foods in the human stomach is
still very vague.

As just mentioned, the stomach is not emptied all at once. It begins
to be emptied very soon after the beginning of digestive activity. Thus
when a dog is fed with meat, the first products of digestion appear in the
duodenum after a few minutes. The stomach is emptied intermit-
tently.^ In feeding 100 grams of meat to a dog weighing 7 to 8 kilograms,
all the chyme was emptied in the course of 2^ hours. It is difficult to get
a correct idea of the time spent by the food in the stomach from such
experiments. They are often very contradictory. We shall understand
immediately why this is so, when we are told that Pawlow has proved
that normally the opening and closing of the pylorus are regulated by the
duodenum. If hydrochloric acid, or gastric juice, is constantly intro-
duced into the duodenum through a fistula, a soda solution placed in the
stomach will be retained during the whole course of the experiment. The
period which normally follows the opening and closing of the pylorus

' Concerning the literature, see E. H. Starling: Ergeb. Physiol. (Asher and Spiro) Jg. I,
Abt. 2, 446 (1902). Extensive studies on the functions of the muscles of birds have
been made by Mangold: Pfliiger's Arch. Ill, 163 (1906).

' Cf. Moritz: Z. Biol. 42, 565 (1901). von Mering: Kongress f. innere Med. Berlin,
1877 and Wiesbaden, 1893. A. Hirsch: Zentr. klin. Med. 47, 993 (1892).

3 Cf. Ludwig Tobler: Z. physiol. Chem. 45, 185 (1905).

508 ' LECTURE XXI.

corresponds evidently to the time required by the alkaline juices of the
intestine to neutralize the hydrochloric acid in the chyme. When this
has been effected, then, reflexively, the pylorus is opened and a new
portion of chyme passes out of the stomach. The suitability of such
an arrangement is quite obvious. We shall see that the fertnents of the
pancreas can act only in neutral or alkaline solutions. If now the entire
acid contents of the stomach were to be suddenly emptied into the intes-
tines, then evidently the subsequent digestion would suffer.' In fact, only
moderate amounts of chyme are to be found in the intestines of animals
killed at various times after an abundant feeding. This is particularly
remarkable when we compare the contents of the tightly stretched stomach
at the beginning of digestion with that of the duodenum. The small
portions of chyme as they leave the stomach are evidently at once further
digested and absorbed. To the fat, also, has been ascribed an effect on
the opening and closing of the pylorus. Enough has been said to show
that the emptying of the stomach may take place with different degrees
of rapidity according to the prevailing conditions. On the other hand, it
enables us to understand why such contradictory statements are found
in the literature concerning the time required. Almost all of the early
investigators followed the course of the stomach's activity, by means of a
fistula in the duodenum, in such a way that the chyme on leaving the
stomach, in part at least, passed at once through the fistula opening,
whereby naturally quite unusual conditions were created, leading to quite
uncontrollable changes in the natural processes.

A question which has been much discussed is whether absorption
begins to take place in the stomach. At present we can only answer this
question in so far as we know that it is certain that the mucous membrane
of the stomach does take up certain substances from the chyme. As soon
as we possess further information concerning the amount and nature of
the absorbed substances, we shall be able to close up this gap in our
knowledge. Pui-e water is not absorbed perceptibly; but, on the other
hand, aqueous solutions of sugar and peptone and of salts lose part of the
dissolved substance and part of the water while they are in the stomach.
The absorption of digested albumin in the stomach has been studied par-
ticularly carefully, but without its being possible to get any clear idea as
to the extent that this takes place. We shall come back to this question
of absorption when we speak of the functions of the remaining parts of the
alimentary canal. We may, however, state in advance that it has not
yet been found possible to refer these processes completely to physical or
chemical laws.

' This fact must be considered in cases where there is an excessive secretion of
gastric juice, and especially of hydrochloric acid. This tends to increase the time
required by the stomach to empty itself.

THE FUNCTIONS OF THE DIGESTIVE ORGANS. 509

Thus far we have concerned ourselves entirely with the stomachs of
man and the carnivora; but, before leaving this part of the subject, we
must pay some attention to a class of animals whose stomachs are much
more complicated, namely the ruminants. The stomachs of these animals
consist of four separate compartments connected with one another. The
food at first passes into the rumen, or paunch, then into the reticulum,
which is connected with the former by means of a wide opening. The
reticulum itself has three openings. One, as just mentioned, leads to
the paunch; a second to the third stomach, which is variously known
as the omasum, psalterium, or manyplies; while the third opening connects
the reticulum, also called the honeycomb, directly with the gullet. The
psalterium provides the connection with the fourth stomach, the so-called
abomasum, or rennet-bag. From the paunch and the reticulum, the food,
which has already been mixed with saliva to some extent, is regurgitated,
or thrown up into the mouth, in from 20 to 70 minutes after it has been
first swallowed. This process is known as rumination, or chewmg the cud.
Each time only certain portions of the food are given up by the stomach.
In the mouth the food is ground up extremely fine and kneaded together
with saliva, after which it is swallowed again, and reaches, if it is already
sufficiently pasty, the psalterium, through the so-called esophageal groove.
The latter leaves one side of the gullet at almost a right angle, and con-
sists of a tube formed by parallel folds communicating directly with the
psalterium. Only pasty and liquid materials can pass along this path.
The solid and semi-solid constituents of the food fall from the gullet
directly into the paunch and reticulum. A part of the food paste also
reaches the psalterium through the narrow passage between it and the
reticulum. In the psalterium the food is still more finely subdivided and
intimately mixed. In the abomasum, the action of the stomach is com-
pleted, and, on the whole, the entire effect is similar to the process which
takes place in other mammals. The first two divisions correspond to the
cardiac portion of a single stomachy and the two latter to the pyloric end.

Finally, we must answer the question whether the stomach is an organ
which is indispensable to life. This is not the case. The entire stomach
has been completely extirpated from a number of dogs and the oesophagus
connected directly with the duodenum without causing any disturbance
in the health of the animal.' Recently the stomach has been successfully
extirpated from human beings a number of times.^ In no case have any
symptoms developed which would indicate that the stomach is an organ

' Czerny: Beitrage zur operativen Chirurgie, Stuttgart, p. 141 (1878). M. Ogata:
Arch. .\nat. Physiol. 1883i 89. Carvallo and Pachon: Arch, de physiol. 5 s^rie T.
6, p. lOG (1894).

^ Langenbuch: Deut. Med. Wochschr. 1894, No. 52. C. Schlatter: Korrespondenz-
blatt Schweizer Aerzte 27, 705 (1897).

510 LECTURE XXI.

indispensable to life, but these operations enable us to understand exactly
what the duty of the stomach is in the economy of the whole organism.
The stomach enables us to partake of our daily food within a relatively
short time and at considerable intervals. It is to a certain extent a
store-room. The stomach is also to be regarded as serving to protect the
intestines. It prevents the injurious action of too hot or too cold foods.
If the stomach has been removed, it is necessary to eat the food in small
portions and at frequent intervals. The food must then be in a pasty
condition before it is swallowed. It is interesting to find that if but a
small piece of the walls of the stomach remain in the system after the
operation, this enlarges and develops into a new stomach, which performs
the functions, to some extent at least, of the original stomach.

LECTURE XXII.
THE FUNCTIONS OF THE DIGESTIVE ORGANS.

II.

From the stomach the food reaches the duodenum, and undergoes an
energetic digestion. Here, as we have ah'eady repeatedly stated, the food-
stuffs which in their composition are comple.x and unlike, as well as entirely
unsuited for direct absorption by the tissues, are to a greater or- less extent
broken down into their simpler components. Thus complicated carbo-
hydrates are transformed into the simplest sugar, the albumins into
amino acids and polypeptides, and the fats eventually into fatty acids
and glycerol. From these materials the body is able to construct the
components of its tissues. Digestion serves not only to make the sub-
stances suitable for absorption, but, above all, for assimilation.

By means of this breaking down of the foodstuffs, the animal organism
makes the cells of its tissues to a large extent independent of the nature of
its food. It is to the cells a matter of indifference whether the food is of
animal or vegetable origin; they will in all cases receive the same carbo-
hydrates, the same fats and proteins from the blood. We may state in
advance that evidently the walls of the intestine themselves play an
important part in effecting the transformation of the separate foodstuffs.
Within them takes place, according to our present knowledge, the building
up of albumin and fat from the more simple components. Absorption
takes place without doubt in proportion as this synthesis is accomplished.
This fact makes it difficult to trace the exact relations of the foodstuffs
to the intestine. Our knowledge ceases essentially with the taking up of
the food by it. It might be thought that some idea of the complicated
processes taking place in the intestine could be gained by causing, with
suitable methods^ an accumulation of the decomposition and synthetical
products, e.g., in the examination of a surviving intestine. Up to the
present time, however, such experiments have failed to give satisfactory
results. The absorption of fats and their synthesis from the simple com-
pounds has alone been followed to a certain extent by means of the micro-
scope. With the proteins the relations are far more complicated. The
walls of the intestine themselves consist chiefly of albumin. It is hard
to tell what is new and what was originally present. As long as we are
unable to differentiate sharply between the different albumins, there is
practically no prospect of our being able to get direct proofs by means of

511

512 LECTURE XXII.

the paths already trod. We must not forget, moreover, that the action of
the ferments, especially those of the cells, is extremely sensitively regulated.
It is entirely dependent upon certain definite external conditions, such
as, for example, the concentration relations. Every disturbance in this
direction must force the entire course of the cell-work into other channels
and quickly bring it to a halt. It is very important that digestion should
be considered, at present, in a broad sense rather than to attempt to
establish its significance in any definite direction. Only from this
standpoint are we able to comprehend the nature of digestion in its com-
pleteness, and from thence new ways and new goals appear for future
investigation in this infinitely complicated field.

In the duodenum, the chyme first comes in contact with the alkaline
intestinal juices. The acid in the food mixture begins to be neutralized.
This juice is partly obtained from glands placed in the mucous membrane
at the beginning of the duodenum, and known as Brunner's Glands; but
the so-called Liebcrkuhn's Glands are more important. These are found
in the mucous membrane of the entire small intestine. Even in the large
intestine similar little glands are found, although these differ in the nature
of the epithelium and in their functions from the corresponding glands
in the small intestine.

The glands of Brunner have been considered by some as small pan-
creatic glands, while others have regarded them as similar to the glands
in the pyloric region of the stomach. The work of Pawlow and Parast-
schuk ' makes it seem probable that these glands yield a ferment which
corresponds to pepsin. These investigators have shown, moreover, that,
like pepsin, this ferment requires hydrochloric, or some other acid, to acti-
vate it. The secretion from these glands also has a milk-coagulating action.
Here, in the same way as with regard to the juice of the pyloric region of
the stomach, it is possible to show that Pawlow's assumption is correct.'

The secretion from the glands of Lieberkuhn has been the object of
much careful investigation. It may be studied by the aid of a fistula,
made in the small intestine. It has been shown that starving dogs pro-
duce no secretion, or at least but very little. Secretion sets in when the
intestine is in any way irritated, whether by mechanical, chemical, or elec-
trical means. Ingestion of food also causes the secretion. The secretion
varies in different parts of the intestine. In the upper part it is less abun-
dant than in the lower. The juice of the intestine reacts alkaline, and
always contains sodium chloride and carbonate in apparently quite con-
stant proportions. According to recent investigations, it contains a fat-
splitting ferment,' and one with a slight amyoh/tic action. Furthermore,

» Z. physiol. Chem. 42, 415 (1904).

' Abderhalden and Rona: Z. physiol. Chem. 47, 359 (1906).

s W. Boldireff: Zentr. Physiol. 18, 460 (1905).

THE FUNCTIONS OF THE DIGESTIVE ORGANS. 513

an invertase, a maltase, and, in mammals, a lactase, have been found.
Finally there is erepsin, already described, which has no direct action
upon native proteins, with the exception of casein, but does act upon
their hydrolytic decomposition products, the peptones.

The function of the mucous membrane of the small intestine is by no
means restricted to the production of the intestinal juice. We shall
soon see that substances are secreted by it which are of far-reaching
importance for the functions of the pancreas and its ferment, trypsin.

The secretions produced by the Brunner and Lieberkuhn cells are un-
important compared with that of two more important accessory glands,
the liver and pancreas. To be sure, this is not necessarily true of the
physiological functions themselves, which in no case are to be judged
entirely from the standpoint of quantity, but rather from that of quahty.
Particularly the more i-ecent investigations have taught us that no matter
how insignificant the function of any organ may appear, it must not be
disregarded. All sorts of different processes are closely related to one
another and influence each other reciprocally. Whether the particular
link in the chain of the total processes is long or short is immaterial.
We may well imagine that the secretion produced by the glands of Brunner
is in many cases highly significant for the chgestion of proteins. The
pancreatic juice is not able, or at least only imperfectly, to attack con-
nective tissue, for example, while pepsin in the presence of hydrochloric
acid quickly accomplishes this. Now we know that when fat is present in
the food the secretion of the gastric juice becomes considerably diminished,
and it is very probable that in such cases the secretion from these intestinal
glands is of great assistance.

One of the above organs, the liver, constantly gives up a peculiar secre-
tion, the bile, which is continually passing through the bile-duct into the
intestine. It should be mentioned at once that the formation of the bile
is continuous, although the amount secreted varies. It continues during
fasting, though in diminished amount. After eating, the secretion in-
creases in amount; and, in fact, it has been found that the extent of the
secretion depends, in part, upon the nature of the food. We shall soon
come back to these relations.'

The bile, as it reaches the intestine, represents a mixture of the secre-
tions of the liver-cells, the glands of the gall-bladder and the biliary pass-
ages. The latter j^ield mucous chiefly. The bile reacts alkaline to litmus.
Its color varies in different animals and likewise in different individuals
of the same species. In man the fiesh bile is usually a golden yellow,
but sometimes it has a greenish hue. It contains, besides salts, mucin
and water, its own specific substances. These are the bile-acids, which
are combined with alkali, and the bile- pig merits. There are also constitu-

' BarMra: Bull, della szienz. med. di Bologna (7) 9, (189S).

514 LECTURE XXII.

ents which are found in other parts of the body as well. These are chole-
sterol, lecithin, soaps, neutral fats, and urea. Conjugated glucuronic acids
have also been detected in the bile. The salts of the bile-acids are of
chief interest to us. We shall come back to the bile-pigments when we
come to consider the pigments of the blood from which the former result.
That the bile-acids owe their formation unquestionably to the activity of
the liver, is evident from quite a number of observations. If, for exam-
ple, the liver be entirely extirpated from a frog, no more bile-acids can be
detected subsequently in its tissues. If they were produced by other
organs, the acids would be formed after the removal of the liver, unless
it is to be assumed that the liver acts in conjunction with other organs in
their production, and that it produces either the original stages or at
least some essential stage in their formation. Although we are compelled
to assume the existence of such reciprocal relations in a great many
cases, still at present it may be regarded as proven that the liver is the
sole place in which these bile-acids are formed. Any other assumption
would appear less probable. In dogs also, it may be shown that the
preparation of the bile is a function of the liver-cells. If the bile duct
is ligated, there is first of all an accumulation of bile. Certain con-
stituents of the latter then pass into the lymph, and are carried by means
of the thoracic duct to the "blood. Now if the thoracic duct be ligated as
well as the bile duct, no more bile-acid can be detected in the blood.

The bile-acids belong to two groups; namely, the ghjcocholic and tauro-
cholic groups. The members of the first group contain carbon, hydrogen,
and nitrogen, and by their hydrolysis yield glycocoll and a non-nitroge-
nous acid; while those of the other group contain sulphur in addition to
the above elements, and on hydrolysis they yield taurine, and similarly a
non-nitrogenous acid. The constitution of the nitrogen-free acid which is
contained in both groups of bile-acids, and which, moreover, appears to
have a different composition in different bile-acids, has not yet been
fully explained. In general, such an acid is known as cholic or cholalic
acid. We will merely mention the fact that the acid has been assumed
repeatedly to be related to cholesterol, but this relation has never been
established satisfactorily.

The relative amounts of these two groups of acids vary according to
the species of animal, and in fact one or the other group may be missing.
Ghjcocholic acid, C26H43NO6, is always present in human- and ox-bile.
Besides this a second acid, gh/cocholeic acid,' is frequently found which
differs from the first with regard to the " cholalic acid " which it yields;
in this case the acid is known as cholcic acid. The solubility relations of

' V. Wahlgren: Z. physiol. Chem. 36, 556 (1002). O. Hammarsteu: ibid. 43, 109
(1904). H. P. T. Oerum: Skandin. Arch. Physiol. 16, 273 (1904). C. Guudelach aud A.
Strecker: Aun. 62, 205 (1847).

THE FUNCTIONS OF THE DIGESTIVE ORGANS. 515

this last acid are different from tiie first cholic acid, and it has a higher
melting-point. A hyoglycochoJic acid ' has been isolated from the bile of
pigs.

Taurocholic acid - is found in the bile of man, carnivora, oxen, and a few
other herbivora, and yields, on being boiled with acids or alkalies, taurine
and cholic acid. It has the empirical formula C26H45NSO7. In the bile
of the goose the so-called cheno-taurocholic acid is found.^

In the bile of the shark, Scijmnus borealis, Olof Hammarsten,* to
whom our thanks are due for most careful investigations concerning
the bile of different species of animals, found in place of the usual
bile-acids, two ethereal-sulphuric acids which he called sci/mnol-sid phuric
acids. They yield on hydrolysis, besides sulphuric acid, a non-nitrogen-
ous acid, scijmnol, which gives the characteristic color reactions of cholic
acid.

As we have said, only one constituent of the bile-acids is understood
in each case as regards its composition, and this is either glycocoll or
taurine. These two substances originate, as we have already discussed
in detail, from the proteins, and, in fact, glycocoll is recognized as one of
the direct cleavage-products of albumin, while it is perfectly evident that
taurine is formed from cystine.

The constitution of cholic acid is not yet clearly established. Mylius '
has succeeded in obtaining from it a monobasic hydroxy-acid with one
secondary and two primary alcoholic groups. By oxidation of cholic
acid, the so-called dehijdrocholic acid C24H34O5 is formed; while by more
energetic oxidation bilianic acid C24H34O8 is obtained, perhaps more
correctly a mixture of bilianic and isobilianic acids. By the oxidation of
bilianic acid, the so-called cilianic acid, C20H28O8, is formed. On reduc-
tion, cholic acid yields desoxycholic acid, C24H40O4. According to our
present knowledge, we can give to cholic acid the following formula:

fCH(OH)
CzoHsi \ (CH20H)2
[COOH

' Severin Jolin: Z. physiol. Chem. 12, 512 (1888); 13, 205 (1889).

' Concerning the preparation of pure taurocliolic acid, see O. Hammarsten, Z. physiol.
Chem. 43, 127 (1904), and Stefan Tengstrom: ihid. 41, 210 (1904).

' Heintz and Wislicenus: Poggendorff's Annal. 108, 547 (1859).

' Z. physiol. Chem. 24, 323 (1898).

5 Cf. Strecker: Annal. 65, 1 and 130 (1848); 67, 1 (1848); 70, 149 (1849). F. Mylius:
Ber. 19, 369 and 2000 (1886); Z. physiol. Chem. 12, 262 (1888). P. T. Cleve: Compt.
rend. 91, 1073 (1880). Olof Hammarsten: Ber. 14, 71 (1881). Lassar-Cohn: ibid. 32,
683 (1899). Z. physiol. Chem. 17, 607 (1893). Fritz Pregl: Pfliiger's Arch. 71, 303
(1898); 72, 266 (1808) ; Sitzber. kaiserl. Akad. Wissensch. in Wien, Math, naturw. Klasse,
111, Abt. II b. October, 1902, and Z. physiol. Chem. 45, 166 (1905). G. Bulnheim:
Z. physiol. Chem. 25, 296 (1898).

516

LECTURE XXII.

Choleic acid has also been oxidized, but this has not served to ex-
plain its constitution. We may mention, in passing, that a cholic acid
described as fellic acid ' C23H40O4 has been obtained from human
bile. In the polar bear another cholic acid has been isolated by
Hammarsten^ which he designated as ursocholeic acid, C19H30O4 or
C18H28O4.

Our present knowledge does not tell us much concerning the for-
mation and destiny of these cholic acids in the animal organism, and
we are forced to rely upon assumptions. It is indeed perfectly possible
that they are related to cholesterol, although this has never been estab-
lished positively.

The composition of the bile varies not only in amount but also as regards
its composition. We shall give a few figures showing the relative amounts
of the different constituents. In 1000 parts of bile from the hepatic duct
there were found to be present : ^

Solids

Water

Mucin and pigments .
Alkali bile-salts . . .
Taurocholates ....
Glycocholates ....
Fatty acids from soaps

Cholesterol

Lecithin

Fats

Soluble salts

Insoluble salts ....

25.40
974.60
5.15
9.04
2.18
6.86
1.01
1.50
0.65
0.61
7.25
0.21

Of the inorganic salts present, sodium chloride predominates. Sulphates
and phosphates are present only in small amounts. The bile from the gall-
bladder shows a somewhat different composition from that taken directly
from the bile-ducts. This is due to the fact that while the bile remains
in the bladder it becomes thickened, owing to the absorption of some of
the water, while at the same time mucin and other substances are given up
by the mucous membrane of the bladder. The following analyses show
the difference in the percentage composition of the bile from these two
sources : *

' G. Schotten: Z. physiol. Chem. 11, 268 (1887). Lassar-Cohn: Ber. 27, 1339
(1894).

' Z. physiol. Chem. 36, 525 (1902).

' Olof Hammarsten: A Text Book of Physiological Chemistry (Mandel), 1908, p. 327.
Cf. also Ergeb. Physiol. (Asher and Spiro) 4, 1 (1905), and Z. physiol. Chem. 32, 435
(1901).

* Olof Hammarsten: NoTa acta. Reg. Soc. Upsal, Serle III, 1894.

THE FUNCTIONS OF THE DIGESTIVE ORGANS.

517

Solids

Water

Mucin and pigments . .
Alkali bile-salts . . .
Taurocholates . . . .

Glycocholates

Fatty acids from soaps

Cholesterol

Lecithin

Fats

Soluble salts

Insoluble salts . . . .

Liver-bile.

bladder.

2.06

16.02

97.94

83.98

0.28

4.44

0.85

8.72

0.11

1.93

0.74

6.79

1 06

0.08

0.87

0.03

0.14
0.15

0.80

0.30

0.02

0.24

The part played by bile in the animal organism has been variously
estimated at different times. Some have even regarded it as an excre-
tion. According to this view, the bile serves merely to carry away the
by-products which are formed as a result of the activity of the cells. We
know that the liver plays an important part in the metabolism of the
animal organism. Important decompositions and syntheses are con-
stantly taking place in its cells. Its position between the blood-vessels
of the viscera and those of the rest of the body make it easy to recognize
its numerous important functions. We have spoken of the position it
occupies with regard to carbohydrate metabolism, and have seen that at
one time the liver cells construct glycogen from glucose molecules, while
at another time it decomposes the latter into its constituents. Many
discoveries indicate that the liver plays an important part in the trans-
formation of fats and proteins into carbohydrates. Even in the metab-
olism of albumin it assumes a central position. In it is effected the
formation of urea and of uric acid. The liver captures the ammonia set
free in the alimentary canal and in the intestines themselves in order to
make use of it in various ways, partly for the formation of urea, and partly
for the neutralization of acid. The liver also stores up many substances
injurious to the organism, or at least foreign to it. This is shown by the
large amount of iron which accumulates here when large quantities of
this element are taken into the system, and by the fact that many other
substances are found in it which are otherwise foreign to the organism.
The liver also effects the combination of many substances injurious to the
system with sulphuric acid and glucuronic acid. It is indeed possible
that in these processes, which by no means include the entire functions
of the liver, waste products are constantly being formed which the cells
of the liver no longer have any use for, and are consequently given up to
the outside. This idea is supported by the fact that certain substances
found in the bile are not indifferent to the organism. Thus we know that

518 LECTURE XXII.

the salts of the bile-acids lessen the frequency of the pulse. This is due to
their action upon the heart. The latter is first of all stimulated, and for
a short time there is an acceleration of the heart action, which, however,
soon becomes retarded. Respiration also becomes less frequent. Thus
we found in describing icterus, which results from the restricted discharge
of the bile into the intestines, what severe pathological conditions appear
if the secretions of the liver-cells are compelled to be eliminated through
the kidneys, being carried thither by means of the lymph and blood-vessels.
The fact that the bile undoubtedly plays an important part in digestion
does not necessarily contradict any such assumption. There may be some
adaptation here. It would not be altogether remarkable if we should find
that the animal organism makes use of a definite function for different pur-
poses. The secretion of bile does not necessarily assume a peculiar position
because it has not yet been possible to find secretory nerves which govern
the secretion. The liver behaves in this respect like the kidneys. The
secretion of bile, according to this, is to be compared to the formation of
the urine. In the case of all the other glands that we have studied up to
this point, we have found secretory nerves. We should not, however,
lay too much stress upon the fact that we have never found any nerves
which in any way govern the secretion of the liver, for it was but a
short time ago that such nerves were positively proved to exist for the
stomach and pancreas. It is not altogether impossible that such
nerves will be found in the case of the liver and possibly for the kidneys
as well. Certain contradictory observations, and much that is not in
accordance with the theory at present accepted concerning the secretion
of the bile and urine, will at once become explicable if nerves can be
found governing the action of these organs.

The experiments of Pawlow and his school have lirought forward many
observations showing the close relation between digestion and the secre-
tion of bile, so that we are obliged to regard bile in the light of a specific
secretion of the liver-cells. The constituents of bile are by no means
waste-products of cell-metabolism, but they are much rather to be regarded,
according to their formation and their entire functions, as true secretion
products. It is indeed possible that the formation of the bile is accom-
plished by means of definite cells. It is also conceivable that their for-
mation is, nevertheless, bound up with the intermediate metabolism in
the liver to the extent that the cells of the liver utilize in a specific way
certain decomposition products. The fact that the flow of bile is con-
tinuous is not contrary to any such hypothesis. Our knowledge concern-
ing the destiny of the bile is still very limited. We do not know whether
a part of it is constantly being resorbed. In fact, frequently the biliary
cycle has been spoken of under the assumption that bile is constantly
being resorbed and again secreted. It has also been observed that the

THE FUNCTIONS OF THE DIGESTIVE ORGANS. 519

bile and its constituents, namely the hile-salts, accelerate the secretion of
bile. Experiments carried out in this direction leave it uncertain for the
present as to what significance we shall give to this resorption process.

Since it was not known precisely what role the bile played in diges-
tion, it has been assumed to be quite different by different scientists.
Some have held that the bile had an antiseptic action. It had been
observed that animals with a biliary fistula showed increased putrefaction
in the intestines. Direct experiments upon the bile itself have shown that
it indeed tends to restrain the action of certain bacteria, but that it is by
no means a good antiseptic agent. I^urthermore, if fat be entirely excluded
from the food, or onl}' a limited amount of it given to animals with such
a fistula, the intestinal putrefaction is not greater than under normal
conditions. It was, therefore, not so much the absence of the bile that
caused the observed putrefaction, but rather the faulty absorption of the
fats.

Bile has, further, been thought to have an influence upon the peristalsis
of the intestines. The extent of such action, under normal conditions, is
still undecided. It has also been observed that if bile is added to a peptic
digesting fluid a precipitate will be formed at once. Now normally the
acid chyme passes out of the stomach mixed with pepsin into the duode-
num. It might be thought that the action of the pepsin, which is no
longer desired in this part of the intestine, is stopped by the bile precipi-
tating pepsin with the albumin and its higher cleavage-products. It has,
however, never been possible to establish satisfactorily the formation of
such precipitates in the intestines, so that at present we are hardly justi-
fied in assuming that this test-tube experiment represents the normal
condition. At the same time it does seem probable that the bile prevents
the further action of the pepsin.

We have already touched upon one quite essential function of the bile,
namely its role in the absorption of fats. We have seen that a large part
of the fats, or perhaps even all, is decomposed into its components the
fatty acids and glycerol. The bile is an excellent solvent for these fatty
acids and the soaps (the salts of these acids), and on account of this
fact a great importance has been ascribed to the bile in assisting the absorp-
tion of the fats and their cleavage-products. Before this action of the bile
had been verified by direct experiment, it had been observed that if, for
any reason, the flow of bile into the intestine was prevented, the fceces
showed a pronounced pale color, and when a considerable amount of fat
was present in the food it was at once obvious that undigested fat was
present. Other facts indicate a faulty absorption of the fats in such cases.
In animals with biliary fistula, where the bile was taken away, animals
decreased rapidly in weight, although fed with the same nourishment
that had previously agreed with them. It is clear that the loss of a

520 LECTURE XXII.

material as rich in caloric power as the fats will be quickly felt through the
entire organism. If the nourishment of the just-mentioned animals was
so chosen that they obtained sufficient calories from albumin or carbo-
hydrates, then there was no further loss in weight. The absorption of
the fat in such cases is not entirely prevented, but merely restricted.
That an excessive amount of fat in the nourishment can act injuriously
upon the absorption and digestion of albumin is clear, for the fat particles,
from their mechanical action, can prevent, or make more difficult, the
action of the digestive juice upon the proteins.

The bile not only takes part in the solution of the fatty acids and their
alkali salts in the absorption of the fats, but its significance reaches much
farther. We must remember that the fat is hydrolyzed l^y the aid of a
particular ferment, called steapsin or lipase. This ferment is not present
originally in the pancreatic juice as such, but in the form of a zymogen.
The latter is activated Ijy the bile. The bile also increases the fat-splitting
action of the pancreas-lipase in a way which has never been satisfactorily
explained.' Fiirth and Schiitz ^ have proved that the cholic acid com-
ponent of the bile salts is the cause of this marked acceleration in the
digestion of fats.

An exact idea concerning the conditions governing the secretion of the
bile and its function was first made po.ssible when Pawlow ^ instead of
making use of a biliary fistula placed the entrance of the bile-duct
into the duodenum on the outside of the body. He cut out the mouth
of the bile-duct together with a piece of the intestinal membrane and
sewed it into the wound in the body. He was then able to study the
secretion of the bile under normal conditions. The observations made
upon animals in which the bile flowed out of a fistula in the gall-bladder
naturally could^ not represent normal conditions, for the bladder repre-
sents a reservoir for the bile, so that when it is constantly being emptied
to the outside, the secretion of the bile must take place abnormally.

Pawlow succeeded in showing at once from the amount of bile flowing
through this normal opening that the bile secretion depended upon the
taking of food. He also showed the influence of the nature of the food
upon the .secretion, i Thus we know that meat causes a particularly in-
tense flow of bile, while for the carbohydrates a slight amount suffices.
The fats stand intermediate between meat and carbohydrates. The
nature of the .secretion is also regulated by that of the nourishment. The
maximum flow of bile does not correspond to the maximum amount of
food, and it appears that there are specific differences here. The purpose

' M. Nencki: Arch, exper. Path. Pharin. 20, 367 (1885).

' Zentr. Physiol. 20, 47 (1906); Hofnieister's Beitr. 9, 28 (1906) — cf. A. S. Loeven-
hart and C. G. Souden: J. Biol. Chem. 2, 415, (1907).

' Pawlow: Le travail des glandes digestive (Pachon et Sabrazes). Paris, 1901.

THE FUNCTIONS OF THE DIGESTIVE ORGANS. 521

of this adjustment to the different foodstuffs is easy to understand,
when we point out that the bile assists the action of the pancreatic juice
by accelerating the action of the ferments. It affects the fat-splitting
ferments most, but it also increases the action of trypsin and diastase.
BUe, consequently, is concerned not alone with the digestion of fat, but
influences considerably that of the other foods. It assists the transference
of the seat of digestion from the stomach to the intestines, probably by
preventing the further action of pepsin.

The functions of the bile are not, in general, considered to be as impor-
tant as would seem probable from the researches of Pawlow. It has been
found, in fact, that the bile may be entirely excluded from the intestines
without any serious disturbance taking place, provided the food be prop-
erly chosen. It would be wrong to conclude from this that the bile is
of little importance in the digestive process, for even with complete extir-
pation of the pancreas, digestion does not cease entirely. The animal
organism possesses ways and means of replacing, to some extent at least,
lost functions. The ferments of the pancreatic juice can indeed perform
their work without the aid of the bile, but it requires more time. Even
then a part of the fat will be absorbed. There are no exact experiments
to inchcate the effect of the loss of the bile upon the metabohsm as a
whole. Its absence is not without influence, because it is then necessary
to choose the food more carefully. Food rich in fat must be avoided.
We merely wish to emphasize the fact that it does not follow because an
animal is able to exist without a certain function, and even be kept in
good health, that the function is perfectly dispensable. Thus we should
make a serious error if we reasoned that because a man could live without
a stomach that this organ occupies physiologically a subordinate posi-
tion. We must accustom our.selves to have in mind the working together
of all the organs, and never follow the functions of a single organ only
under certain special conditions but under as many different conditions
as possible, and especially under those which occur normally. Only in
such cases are we able to form a proper judgment as to the relative value
of the functions of a given organ.

Now that we have traced the transition between the digestion of the
stomach and that of the intestines, we must turn our attention especially
to the functions of the pancreatic juice. This contains, as we have stated
repeatedly, three ferments, — trypsin, steapsin, and a ferment which has
a diastatic action. While it has not yet been established positively that
the last ferment is secreted originally in a zymogen condition, this is surely
the case with the two other ferments, trypsin and steapsin. Trypsinogen
is, according to the important observations of Pawlow, activated by a
substance which occurs in the intestinal juices. Pawlow called this
substance enterokinase. It is very likely that this substance itself belongs

522 LECTURE XXII.

to the group of ferments. It is possible to activate, by means of very
small quantities of enterokinase, large amounts of trypsinogen. It is
evidently to be considered as a secretion of the intestinal membrane.
Intestinal juice, obtained through a fistula, may be added directly to
the inactive pancreatic juice. A preparation of enterokinase may also be
obtained by scraping off the superficial layers of the intestinal membrane
and preparing an extract from these scrapings. The action of the entero-
kinase may be well shown by taking some pancreatic juice which has never
been in contact with the walls of the intestine, so that the ferments con-
tained in it are inactive,' and placing some fibrin in it which will not dissolve.
Now if we add to another portion of the same juice a few drops of intestinal
fluid, or of the extract prepared from the intestinal walls, it will be seen
that a piece of fibrin in it dissolves at once. It is not yet perfectly clear how
this enterokinase acts. It might be even assumed that there is not a true
activating in this case, but that the enterokinase is itself a proteolytic
ferment and begins the cleavage of albumin. We have already seen that
pepsin attacfe the albumin molecule in an entirely different place than
does trypsin. It is possible that the enterokinase continues the work
of the pepsin and gives up cleavage-products to trypsin which the latter
is capable of acting upon. We might also assume that enteroldnase
attacks trypsinogen itself, modifying it in some way so that trypsin now
has certain groups free whereby it can react with alliumin, or its cleavage-
products. One might lie tempted even to assume that between trypsino-
gen and enterokinase a union takes place and that active trypsin is
thereby formed. If this assumption were correct, then of course the entero-
kinase and trypsinogen must always be active in quite definite proportions.
This does not appear to be the case, however, for it is possible by means of
but very little enterokinase to activate a great deal of trypsinogen. We
have here again a reciprocal effect of different organs. The pancreas
sends out a zymogen, and the cells of the intestinal membrane form the
activator. Pawlow and his student Sawitsch ^ have shown b}^ very pretty
experiments that the secretion of the intestine does not invariably contain
enterokinase, whether the pancreatic juice reaches the intestine or not. If
a canula be introduced in an intestinal fistula, then this mechanical irrita-
tion causes a secretion of intestinal juice. Onlj^ a small amount of entero-
kinase is present in this juice, which consists chiefly of water. In the
course of a few hours the intestinal fluid, which is now scanty in amount,
contains almost no enterokinase. If now a few culiic centimeters of
pancreatic juice be introduced into the intestinal canal, then a juice flows

' It should be mentioned here that apparently the ferments in the pancreatic juice
always contain a small amount of trypsin in the active form. Cf. B. P. Babkin: Ber.
kaiserl. Militararztl. Akad. St. Petersburg 11, Nos. 2 and 3, 93 (1904).

2 W. Sawitsch: Soc. mdd. russes St. Petersburg (1900-01).

THE FUNCTIONS OF THE DIGESTIVE ORGANS. 523

out which is rich in enterokinase. Boiled pancreatic juice, however, has
no such effect. The secretion of the intestinal juice is, according to this,
by no means such a simple process as has ordinarily been assumed. Its
composition is determined by at least two factors which are largely
independent of one another. The production of enterokinase, and the
formation of the other constituents of the secretion produced by the
intestinal membrane and its glands, are distinct processes. The intestinal
juices have a favorable action upon the digestion of albumin, not only
by reason of the enterokinase, but, according to many observations, also
on account of the other ferments in the pancreatic juice. These juices
have on the whole a quite similar effect to the bile.

In describing the secretion of the stomach, we saw that the amount and
composition of the gastric juice are dependent upon various external
influences, and that above all psj'chic influences play an important part.
Is the secretion of the pancreatic juice similarly affected? The following
observations give us some light with regard to this important question.
In the case of herbivora in which the digestion is, so to speak, a continuous
process, the secretion of the pancreatic juice takes place continually. In
carnivora, it is possible to trace at once some connection between the
introduction of food and the subsequent digestion.

Pawlow called attention, in the first place, to the following experiment:
If a few drops of 0.5 per cent hydrochloric acid are introduced into the
stomach of a dog having a pancreatic fistula from which only a few drops of
pancreatic juice are flowing in a minute, there is then an increase in the
secretion after a short time. If instead of acid a little lime-water is intro-
duced into the dog's stomach, the contrary effect is obtained. Phosphoric,
lactic, citric, and acetic acids each have the same effect as hydrochloric
acid. The concentration of the acid, moreover, greatly influences the
secretion, as the follomng experiment shows:' 250 cubic centimeters of HCI
of the follomng concentrations were introduced into the stomach of a dog:

o.sTf

O.lTo

0.05':;

Cubic centimeters of pancreatic

juice se-

70.8
79.5
82.5
89.4

25^7
26.8
32.5

20.5

The pancreatic gland reacts promptly with the acid, and even with
concentrations which bai-ely have an acid taste. Other irritants, such as
pepper and mustard, are without influence. Acid, therefore, is to be
regarded as exerting a specific effect upon the pancreas. Naturally the

Pawlow: Vorlesungen etc., op. cit. p. 150.

524 ' LECTURE XXII.

gastric juice has the same effect as a pure acid solution of the corresponding
concentrations. The following experiment is important: If soda or lime-
water be introduced into the stomach of an animal in the midst of the
process of digestion, there is a rapid diminution in the amount of secretion
from the pancreas.

We have here a new link in the chain representing the mutual dependence
of one organ upon another. The pancreas regulates its activity according
to that of the stomach, and is governed chiefly by the acid produced in the
latter. The next question that arises is how the hydrochloric acid of the
stomach effects the stimulation of the pancreatic gland. There are two
possibiUties. It may be that the acid stimulates the peripheral end-
apparatus of the centripetal nerves in the mucous membrane, or that it
acts upon the nerve center of the secretory cells of the pancreas, or upon
the cells themselves, after it has been taken up by the blood. The latter
method of action is improbable for a number of reasons. Pawlow showed
that the acid taken up by the blood could only have an indirect action;
namely, by diminishing the alkalinity of the blood. Now, normally, the
alkalinity of the blood is increased by the production of hydrochloric
acid, and even in case of an absorption of hydrochloric acid, it remains
higher than usual during the period of digestion. Direct experiment con-
firms this view, for, on the one hand, it is not possible to stimulate the
secretion of the pancreatic gland by means of introducing hydrochloric acid
into the rectum, while, on the other hand, the action of the hydrochloric
acid is still felt even when its passage out of the stomach is prevented.'

Now how shall we explain the action of the hydrochloric acid? Pawlow
brings out the following points: — Trypsin i-eacts best in an alkahne
solution, but is still active in a neutral or even barely acid solution. As
soon as the amount of acid becomes in any way considerable, the action
of the trypsin is prevented. Now the pancreatic juice always contains an
abundance of alkali by means of which the acid in the chyme is neutral-
ized. The more acid the stomach produces, the more acid reaches the
intestine with the chyme, and the more alkali is required to combat the
injurious effect of the acid. The fact that the secretion of the pan-
creas is governed by that of the stomach tends to equalize the conditions.
If the amount of pancreatic juice secreted were independent of the hydro-
chloric acid in the chyme, then it would often happen that the trypsin
would be made inactive, and the activity of the pepsin, which under normal
conditions is prevented by the neutralization of the acid it requires, would
continue in the intestine. The whole arrangement may be traced in the
cycle of common salt, somewhat as follows : — The cells of the stomach
prepare hydrochloric acid from the sodium chloride in the blood. The

' L. Popielski: Inaug. Diss. St. Petersburg (1896); Zentr. Physiol. 10, 405 (1896);
Pfliiger's Arch. 86, 215 (1901), and Zentr. Physiol. 16, 43 (1903).

THE FUNCTIONS OF THE DIGESTIVE ORGANS. 525

more hydrochloric acid there is produced, the greater becomes the alka-
linity of the blood. This excess of alkalinity i.s given up by the blood to
the cells of the pancreas which employ it in the production of the pan-
. creatic juice. With the pancreatic juice, this alkali, chiefly as sodium
carbonate, flows into the intestines, and meets there the hydrochloric acid
from the stomach. Here again common salt is formed which may enter
into the circulation anew. At the same time, by means of such a mech-
anism the alkalinity of the blood varies only within narrow limits. We
should not, however, imagine that the process takes place in such a simple
form that the cells of the pancreas are immediately brSught into activity
by the increased alkalinity of the blood, which, in turn, is caused by the
production of hydrochloric acid in the stomach. Plausible though such an
assumption may be, it does not correspond with the results of experimental
research. The production of acid does not have such a direct influence
upon the activity of the cells in the pancreas. We must remember that
hydrochloric acid introduced from without, also effects the production
of the pancreatic juice. In the last case the alkalinity of the blood is
diminished rather than increased. Although the above-described salt-
cycle appears to be a very suitable arrangement, it does not, on the other
hand, stand in direct connection with the action of the acid upon the
function of the pancreatic gland. We must, on the contrary, conceive this
to be due to some phenomenon Ijrought about by the action of the acid
upon the membrane.

We shall soon come to a very important observation of Bayliss and Star-
ling which will shed considerable light upon the nature of the action of
the hydrochloric acid.

It is interesting to find that even the composition of the pancreatic juice
is adjusted to that of the acid in the chyme, for, as Walther '- has showed,
the amount of organic material in the former is regulated by the amount of
acid in the latter. The juice produced by hydrochloric acid alone contains
less organic material and more alkali than the normal. Here again we
meet with the same conditions as in the secretion of the gastric and intes-
tinal juices. Likewise the formation of the pancreatic juice is not that of a
simple substance. It must not be thought that the cells of the pancreas
always yield one and the same secretion. At one time the juice is rich in
ferments, and at another time alkali predominates. For the present we do
not know whether this is due to the fact that cells are influenced differ-
ently by various kinds of nervous stimulation, or whether particular cells
are provided with quite definite functions. The fact that even the juice
rich in alkali, which is produced by the action of acid alone, always eon-
tains ferments, makes it seem probable that the action of the individual
cells is governed by the nature of the stimulation it receives, and that it is

Inaug. Diss. St. Petersburg (189G).

526 LECTURE XXII.

hardly right to believe that certain cells produce the ferments while others
merely give up salts.

At all events, in considering the work of digestion, we are constantly
meeting with an extremely sensitive means for regulating the work of the
cells. They do not always act in the same way, but adjust their action
to the prevailing conditions. In considering the functions of the gland-cells
we gain considerable insight into the activity of the cells of the animal
organism in general. We are led to infer that even the individual cells of
the body are to a consideraljle degree dependent upon one another. They
adjust their work in the same way as the gland-cells, to the given condi-
tions. To be sure, it is perfectly possible, and in fact most probable, that
those cells of the body which are not directly connected with the work of
the intestine, are much more regular in their activity than the cells of the
intestine and the associated glands which are constantly meeting with new
conditions. The intestine forms a solid barrier between the heterogeneous
compounds in the food and the homogeneous building-material for the
blood and tissues, the composition of which has been established by the
entire development of the given animal species. The destructive activity
of the digestive ferments, together with the syntheses taking place in the
intestine, enables the cells of the body to work within certain limits always
under the same conditions. At the same time, the greater demands which
are now and then placed upon an organ, influence the cell work quite
specifically.

The activity of the pancreas is not dependent upon the acid content
alone of the food as it reaches the duodenum. It has been found that fats,
likewise, have an effect. We have already seen that such food diminishes
the amount of gastric juice secreted. The secretion of the pancreatic
juice cannot then, as in the case of meats, be influenced by an increased
secretion of hydrochloric acid. Fats, on the contrary, stimulate directly
the secretion of pancreatic juice. This may be shown by means of a dog
provided with both gastric and intestinal fistulas.' If after waiting until
there is practically no gastric secretion, olive oil is allowed to flow into
the stomach, then the slight amount of gastric juice secreted will have an
alkaline reaction. At the same time there will be a marked increase in the
amount of pancreatic juice. It is questionable whether the fats, and the
soaps produced from them, have the same point of attack as the hydro-
chloric acid.^

It has proved very difficult to ascertain whether the secretion of the pan-
creas is influenced by the same chemical sul)stances as that of the stomach.
This could be answered satisfactoril}' only when there was no opportunity
given for the hydrochloric acid itself to exert a stimulation. Under such

' N. Damaskin: Verhand!. Gesellsch. russ. Aerzte, St. Petersburg (189G).
' B. P. Babkine: Arch, des Sciences biol. 11, No. 3 (1905).

THE FUNCTIONS OF THE DIGESTIVE ORGANS. 527

conditions, it was found that even water is to be regarded as a direct
stimulant of the pancreas. The extractive substances from beef, on the
other hand, did not cause anj' stimulation. Quite recently the influence
of alcohol upon the pancreatic secretion has been studied,' and in this
case it was found that, while the amount of the secretion was augmented,
the juice then had less digestive power. Alcohol also appears to have a
direct action upon the ferments or their antecedents. If alcohol is added
to pancreatic juice, then the digestive action of the latter upon starch and
albumin is much lessened, while on the other hand the action upon the
fat-splitting ferment is favorable.

It was highly important to establish the fact that the psychic factor
also played a considerable part in the work of the pancreas. The vagus
provides this organ as well as the stomach with secretory nerves. Further-
more, it is also claimed that the splanchnic sends fibres to the pancreas.
It was extremely cUflScult to determine whether the secretion of the pan-
creatic gland was effected by a fictitious meal, and for the following
reasons: We have seen that the secretion produced by the memlDrane
of the stomach, and its glands, is greatly dependent upon psychic influ-
ences. A subsequent increase in the amount of pancreatic juice secreted
may, therefore, take place on account of the increased acid production in
the stomach; i.e., in this case the pancreas would be merely indirectly
affected by the fictitious meal. Now we know that the secretion of the
stomach does not take place at once, but only after a latent period of about
four and one-half minutes. The pancreatic secretion similarly begins two
or three minutes after it has become stimulated by acid. It was found
that the augmented pancreatic secretion resulted within two or three
minutes after the beginning of the fictitious meal, so that from this the
conclusion may be drawn that the gland is directly influenced psychically.
It is important that the pancreatic gland, in spite of its dependence upon
the other organs, especially the stomach, still has a considerable amount
of independence, so that even in the absence of stimulation from the
stomach, it can perform its functions. Experience gained from day to day
teaches us that if, for example, there is an insufficient amount of hydro-
chloric acid formed in the stomach, digestion is not prevented, but still
progresses to a quite remarkable extent.

We must in adchtion consider a very important discovery for which we
will have to thank two Englishmen, Bayli.ss and Starling.^ They showed
that by means of 4 per cent h3'drochloric acid a substance could be extracted
from the mucous membrane of the intestine which, when introduced into
the circulation, increased the flow of pancreatic juice. They called this
substance secretin. They have assumed that this substance is not present

' A. Gizelt: Zentr. Physiol. 19, 769 (1906).

2 J. Physiol. 30, 61 (1903); Proc. Roy. Soc. 73, .310 (1904).

628 LECTURE XXII.

as such in the intestinal membrane, but that its antecedent, prosecretin,
is there, and becomes changed into secretin on being acted upon by acid;
i.e., it may be set free in this way from some other compound. It might
also be assumed, of course, that the prosecretin undergoes an atomic
rearrangement in the molecule. Now how shall we regard the action of
the secretin under normal conditions? We must remember that Pawlow
found that the hydrochloric acid from the stomach stimulated the secre-
tion of pancreatic juice. We have already shown how hydrochloric acid,
directly or indirectly, by altering the alkalinity of the blood, can excite
into activity the pancreas, and have left it open as to how the acid acts
upon the intestinal membrane. The work of Bayliss and Starling may
perhaps serve to explain how the hydrochloric acid can influence the pan-
creatic gland. Evidently it is constantly changing prosecretin into secretin.
As quickly as the latter is formed, it is taken into the circulation, and
now acts in some way upon the gland. It seems most probable from cer-
tain observations that secretin affects the blood-vessels in the pancreas
and increases the circulation. This does not necessarily imply that there
is not some specific action as well. This would seem quite hkely from
the fact that secretin stimulates only the pancreas to any extent. Now
this fact gives to the hydrochloric acid of the stomach an entirely new
significance. Not only does this aid us in our knowledge concerning the
action of acid upon the intestinal membrane, but we obtain, at the same
time, new prospects for further investigations concerning the cell-work of
the glands and tissues. Even though we may be a long way from being
able to understand the entire chain of processes, from the formation of
the hydrochloric acid in the stomach to the production of the secretion
on the part of the pancreas, and understand the phenomena only approx-
imately, still we are justified in hoping from the work of Pawlow and of
Bayliss and Starling that in following the paths now broken it will not
be very long before one link after another will be added until finally the
complete chain is forged. To be sure, there remain countless problems
to be solved. We should like to know exactly what prosecretin is, and
to what class of chemical compounds it belongs." The fact that it is not
a ferment is shown by its being unchanged by moderate heat. The intes-
tine, therefore, concerns itself not only with the absorption and assimilation
of the food, but takes part to a considerable extent in the digestion itself.
The anatomical evolution of a unit from the intestine and its accessory

' Popielski, Zentr. Physiol. 19, 801 (1906), has recently proved that unquestionably
HCI also reflexively influences the secretion of the pancreas by its action upon the intes-
tinal membrane. He believes that secretin belongs to the group of peptones. If this
be true we have here a case of one of the products of digestion acting upon this pancre-
atic secretion. The discovery of Bayliss and Starling will of course only receive its full
value when it is possible to isolate the secretin, and study by itself the action of HCI
upon it.

THE FUNCTIONS OF THE DIGESTIVE ORGANS. 529

glands, the liver and the pancreas, also to a certain extent corresponds to
the physiological significance. The work of digestion is not entirely rele-
gated to these glands, but the intestines help to a considerable extent.

From what we know concerning the work of the stomach, and the intestine
with its accessory glands, we -can readily understand at how many places
the total work of these organs may be disturbed, and how many disturb-
ances may result from the loss of a single function. Let us imagine, for
example, that the stomach fails to secrete hydrochloric acid. First of all
'the food will not be utilized in the system to so good an advantage. To
be sure, our knowledge of cookery enables us to overcome many such diffi-
culties. If we were compelled to rely upon food in its original condition,
then the effect of the loss of hydrochloric acid would be far more pronounced.
Thus, for example, connective tissue is scarcely attacked at all by trypsin,
while it is readily cUgested by means of pepsin in acid solutions. Thus the
albumins present in such tissue would reach the intestine in an undigested
condition. An increased secretion of trypsin .would be required on account
of the deficient preliminary digestion. The stimulation usually brought
about by means of hydrochloric acid, however, would not take place.
Thus one disturbance follows another. It is not safe to assume that in
such cases the pancreas would entirely fail to undergo any stimulation.
We have seen, on the contrary, that it is a fairly independent organ,
and may be stimulated by fats and by water as well as by psychic
events.

The knowledge of all these mutual relations with regard to the most
varied functions of different organs at once explains the therapeutic
measures that are taken in case of stomach trouble, whether it be on
account of nervous or organic disease. Now we understand how the
so-called stomachics have an effect, and why under some conditions hydro-
chloric acid itself is introduced into the stomach. On the other hand, it
becomes clear to us how cautious we should be with the use of alkalies.
They serve not only to neutralize the gastric secretion, but they also lessen
the secretion of the pancreatic juice. We are now able to view in a clear
light the functions of the intestine in the economy of the animal organism.
It seems to us not at all impossible that a faulty function on the part
of the intestine in any one of its various function? has an influence in
much greater measure than is ordinarily assumed upon numerous patho-
logical processes. Not the least cause of diseases of metabolism is an
anomally in the complicated processes of the intestine. In the intestine
the cleavage-products of proteins, the fats, and certain other compounds,
are again welded together. A faulty synthesis, or a building-up in the
wrong direction, must immediately have its effect upon the general meta-
bolism, for the ferments in the cells are only adjusted to react with quite
definite compounds. We make these suggestions merely to show what a

530 LECTURE XXII. I

deep significance is to be ascribed to the intestine in the general metabolism
of the organism.

We have up to this point merely considered the pancreatic juice as such,
and, with the exception of its alkali content, have paid little attention to
the secretion of its individual ferments. We have seen that trypsin and
steapsin are secreted as zymogens, while for the diastase arguments have also
been advanced, though probably wrongly, in favor of a zymogen condition.
The knowledge of the fact that the two first-named ferments exist in two
states, was of great i-mportance for subsequent investigation, and above
all it was very significant that trypsinogen was activated by a constituent
of the intestinal fluid, namely enterokinase. In introducing pancreatic
fistulse, usuallj' the entrance point of the principal duct from the pancreas
into the duodenum is sought, and then the papillae, together with the piece
of intestinal membrane bearing it, is cut out from the alimentary canal and
sewed into the wound in the body. If the pancreatic juice flowing through
such a fistula be examined, it will be found that it is alwaj's active. This
is due to the fact that the pancreatic juice thus obtained is always mixed
with some secretion from the piece of intestine. If it be desired to obtain
the pancreatic juice in an inactive condition, this little piece of intestinal
membrane must be removed completely.' It has been found that even
such juice, under certain conditions, may contain, besides the zymogens,
active ferments as well. Thus we know that bj^ the introduction of acid
and of soaps into the intestine a juice more or less rich in active ferments
results. Again, in the case of nourishment with a mixed diet, there
is obtained a varying amount of active ferments, the quantity depending
upon the nature of the food.^ When meat is eaten, for example, the
largest amount of zymogens is obtained, while the least amount results
from a milk diet. Bread occupies an intermediate position.

Before the fact was known that the pancreatic ferments are, for the
most part, given up in the form of zymogens, and that these are activated
by the intestinal juices, it was considered as proven that each food caused
the production of all three ferments, but that the fat-splitting ferment
was present in largest amount. As Babkin has shown, this specialization
does not take place. The three principal ferments of the pancreatic juice
are, under physiological conditions, secreted practically evenly. If the
value of the pancreatic juice obtained after eating a certain kind of food
is based upon the amount of proteolytic ferment the juice contains, it is
found that milk produces a secretion of greatest digestive power. The
two other ferments, diastase and steapsin, are likewise present in consid-
erable amount. The activity of these two ferments remains in this case

' B. P. Babkin: Ber. kaiserl. militariirztl. Akad. zu St. Petersburg 9, Nos. 2 and 3,
93 (1904).

' Babkin: ibid. 11, Nos. 2 and 3, p. 93 (1904).

THE FUNCTIONS OF THE DIGESTIVE ORGANS. 531

about the same for several hours. After eating meat, the digestive power
for albumin sinks very rapidh' during the .second hour, onlj' to rise again
considerably above its original value in the following hours. Diastase
and steapsin behave similarly.

Again, the amount of juice depends upon the nature of the food. Bread
produces the most secretion, then follows meat, while milk occupies the last
place.

We must mention, in pa.ssing, the fact that it has been suggested that
the spleen also is related to the secretion of the pancreas, and takes
part in activating the trypsinogen. It has been observed that an extract
made from the spleen, which has been removed during digestion, strength-
ens the action of the pancreatic juice. Pawlow, however, states that he
could not find that the secretion produced from animals with the spleen
missing had less digestive power than from those with the organ intact.'

Under normal conditions, a single foodstuff does not usually come by
itself under the influence of the secretion of the pancreas, and of the walls
of the intestine, but rather a mixture of foods. The relations are further
complicated by reason of the fact that it is not these foods themselves,
but rather their cleavage-products, which are acted upon. For the present,
the effect of this heterogeneous mixture of products cannot be stated. We
can merely assume from the work of Pawlow and his school, performed
imder uniform conditions, that there are a great many ways here in which
the system adapts itself to the prevailing conditions. We have already
mentioned the fact that the acid chj^me from the stomach does not enter
the duodenum in a continuous stream, but that the contents of the stomach
leaves it intermittently in relatively small amounts. These portions are
at once energetically digested. The products formed by digestion are
constantly being absorbed. Even when a very large quantity of food is
eaten, there is never a large amount of chyme in the intestine. The extent
to which the food is utilized depends, as we shall see later on, largely
upon its nature. Naturally the condition of the intestine also comes into
consideration. In case of increased peristalsis, the absorption may be
lessened. The unabsorbed residue, together with the secretions of bile,
pancreas, intestinal membrane and its glands, compose the f;Eces, or stools.
The absorption takes place throughout the entire small intestine, but is
undoubtedly most energetic in the jejunum. We must mention in this
connection erepsin, which, according to Cohnheim, acts like trypsin upon
peptones, and a,ssists in their absorption.

The chyme is carried on its way by peristalsis. The movements of the
intestines are regulated by the central nervous system. Innervation
is provided in part by the vagus and partly by the splanchnic nerves.

a. Oskar Prym: Pfliiger's Arch. 104, 433 (1904).

532 LECTURE XXII.

The latter are said to contain inhibitory fibers. The innervation relations
are, however, not perfectly understood.

We must now turn our attention to the absorption of the digested pro-
ducts. We approach this part of the subject with considerable hesitancy,
for we must admit at the start that we are not yet able to give a full account
of the nature of the absorption process. We can indeed affirm that
undoubtedly physical forces come into play here, and that, for example,
osmosis plays a part, as is obvious from the already-mentioned observations
of Overton on the solubility of lipoids; and similarly we cannot doubt that
the surface-tension is to be regarded as important here, in the sense sug-
gested by Traube.' On the other hand, we are very well aware that none
of the attempted explanations of absorption have of themselves brought
the entire complicated process nearer to our comprehension. As soon as
a single phenomenon in a single process is applied to the entire absorption,
the explanation in all cases appears arbitrary.

We are not able in explaining absorption, to circumvent the conception
of a specific action on the part of the cells. We can indeed believe that
probably a purely physical explanation will account for an inter-epithelial
absorption. The greater part of the products of digestion will, however,
be taken up by the cells themselves, and these are undoubtedly very active
in their work. We cannot imagine, for example, that the amino acids
and polypeptides, which represent decomposition products of the proteins,
penetrate into the cells purely on account of physical reasons without
active cooperation on the part of the cells themselves. We must not
forget that syntheses immediately follows the absorption; i.e., in other
words, the activity of the cells then begins, and, indeed, as the relatively
constant composition of the serum shows, in a quite definite direction.
We have no reason for assuming that in the epithelium of the intestine and
the cells of this organ, certain forces unknown to us are at work. If we
were to make any such assumption, it would be entirely without empirical
justification. Although, at present, we are denied an accurate insight
into the nature of absorption, still on the other hand our knowledge of
the work performed by the cells is constantly increasing. The intestinal

' It would not be difficult with the aid of the H. J. Hamburger's " Osmotischer Druek
und lonenlehre in den medizinischen Wissenschaften " (1902) to cite the diilferent
views held regarding intestinal absorption. On the other hand, it would be hard,
without going into a detailed explanation of the laws and investigations of physical
chemistry, to give a clear picture of the different hypotheses in this narrow field. We
would refer the reader, therefore, to the above book and to the following literature:
Rudolph Hoeber: Pfluger's Arch. 70, 624 (1898); 74, 246 (1899); 86, 199 (1901); 94, 3.37
(1903); O. Cohnheim: Z. Biol. 36, 129 (1897); 38,443 (1899), and 39, 167 (1900); also
Z. physiol. Chem. 33, 9 (1901); 35, 396 and 416 (1902). J. Traube: Pfliiger's Arch.
105, 541 and 559 (1904). Cf. also Martin Heidenhain: Anatomische Hefte, 79-80,
26, 2-3 (1904). Published by Merkel and Bonnet.

THE FUNCTIONS OF THE DIGESTIVE ORGANS. 533

absorption is to be regarded as a process wiiich is no more cojnplicated
than the formation of a secretion. In the latter case cells take away
certain substances from the blood, while in the former case other substances
are taken from the digesting mixture. Just as the cells of the gland show
a selective power, so also those of the intestine have the power of choosing
their material. In considering the action of ferments we emphasized their
specific action and suggested that this is due to the peculiar structure of
the ferment molecule. We can apply the same reasoning to the cells
themselves, and beheve that they are specific in their entire construc-
tion, and similarly are merely able to take up substances having
particular atomic groupings in the molecule. Indeed, we cannot
abandon the thought that the cells of the intestine in a certain sense form
a secretion from the substances obtained from the food which they give
up on the other side of the intestinal wall. As the gland-cells take the raw-
material from the blood for the formation of their specific secretion, and
then in a short time throw it off only to build up more of it, so here we
can imagine that the cells in the intestine carry on their work in a similar
manner, and acting together eventually give to the blood a homogeneous
material. Certain residues are taken up by the lymph where they are
carried first to the mesenteric glands, from thence to be gradually given
up for further metabolism.

It would be absurd to consider absorption to be a result of an unknown
force, merely because we are at present without insight into the process.
It is not without interest in this connection to recall an example which
at first glance appeared to show strikingly an actual intelligence on
the part of unicellular organism, but which can be explained more
simply. We refer to the observation of Cienkowski.' He studied the
absorption of nourishment by the Vampi/rella Spirogyrce. It is a micro-
scopically-small, naked, reddish-colored cell. This simple being, in
which not even a nucleus is discernible, seeks out, among all the various
algce that are at hand, always a certain especial kind, and leaves untouched
all other varieties. When it has come in contact with the suitable kind of
Spirogyra, it places itself firmly next to the cell-wall, dissolves it and
sucks in the contents. We now know enough concerning the action of
ferments, however, to show that the fact that this kind of Vampyrella
feeds only upon special algae is not so remarkable. It is quite certain
that the cell-wall is dissolved by means of a ferment. The ferments are
evidently capable of acting only upon a certain kind of alga. We mention
this example at this place especially to show how we should look upon the
active absorption of substances on the part of the cells. It may be merely

' Arch, mikroskop. Anat. 1, 203 (1865). Cited by Bunge in his Lehrbuch der Physi-
ologie des Menschen, Vol. II, p. 4 (1901).

534 LECTURE XXII.

pointed out, that the cells come into consideration according to the way
that they are constructed, and that evidently chemical processes play an
important part in the phenomena. In no case should it be implied that
forces unknown to us come into play. It is self-evident that we should
recognize clearly just how far we can go in accordance with observed facts
and as to where the realms of pure speculation begin. Unquestionably
we are at present far from understanding the action of the cells. As long
as we do not understand the composition of albumin and especiall}' that
of the ferments, we cannot expect to receive much li'ght upon the numerous
problems which we meet with in studying the work of the cells.

The absorption of the individual foodstuffs, their further destiny in
the tissues and eventual combustion, we have already considered in detail,
so that we will now merely consider one other function of the intestine,
namely, the formation of the fseces and their removal from the system.
We have already found that the amount of excreta varies with the nour-
ishment. The color of the fteces changes similarly. Where an abundance
of meat is eaten the scybala are dark or grayish colored, while a diet
largely of bread makes the color lighter. The bile-pigments have a good
deal to do with the color of the faeces, although it is usually their trans-
formation product, stercobilin, which is present. The fteces contain besides
indigestible substances, the secretion of the intestines and of the accessory
glands, and a certain amount of digestible matter which was not absorbed
for some reason or other. We also meet with products of putrefaction
such as skatole, indole, purine bases, lime and magnesia soaps and other
substances. The iseces furthermore always contain inorganic salts, whether
it be due to the fact that they were not absorbed from the food, or whether
they were eliminated in the intestines.

The formation of the faeces takes place in the large intestine. Here the
unabsorbed material passes, and becomes thickened by loss of water.
Without doubt, in the case of the herbivora. the ferments continue their
action in the large intestine, and utilize for the organism certain amounts
of otherwise undigested material. In the carnivora, however, there is
no digestion worth considering in the large intestine.

We have now mentioned all the functions of the digestive organs. We are
well aware that we have failed to give a clear picture of the total work of
digestion. Still, we are able to take up certain phases somewhat in detail.
On the other hand, so many new vistas in this field have been opened
up by the investigations of Pawlow and his school and of Bayliss and
StarHng, and so many new questions remain to be answered, that we no
longer can have any doubt that the isolated discoveries obtained here
and there will before long be welded together into an organic whole, so
that little by little we shall win more and more from the vast field of
the unknown.

LECTURE XXIII.

THE BLOOD.
Coagulation. Composition.

Blood is the intermediary in the general metabolism. It carries in part
directh- from the intestine, and in part indirectly by the aid of the lymphatics,
the proper nourishment for each individual cell of the body. The oxygen,
which is so indispensable for the work of the cell, is also carried to it by
the blood. On the other hand, the cells give up the products of their
activity, whether as residues from the various combustion processes, or
whether as secretion products which are yet to play an important part
in the total metabolism, to the blood. From all this it is obvious what
a dominating position the blood holds in the animal organism. In contrast
to the other tissues, it is a liquid which is kept in constant circulation by
the action of the heart. The blood always contains numerous cells,
especially the red and the white blood-corpuscles. We have already dis-
cussed the important part that the former play in external and internal
respiration. Besides these form-elements there are blood-plates, the sig-
nificance of which has never been explained satisfactorily. The cell
elements of the blood lie suspended in a liquid rich in albumin, the plasma.
They may be removed from the latter by a centrifugal machine. The
clear yellowish plasma is then obtained above the deposited blood-cor-
puscles. This separation into these form-elements and the plasma can be
effected, however, only under quite definite conditions. If we take blood
from any blood-vessel (usually the carotid is chosen), and simply allow it
to stand, it soon undergoes a peculiar transformation. There settles over
the bottom of the dish containing it a firm coagulum which incloses the
corpuscles. Above this so-called blood-clot there is a clear liquid which is
very similar to the plasma, but is, notwithstanding, an entirely distinct
substance, as we shall soon see. This liquid produced by the contraction
of the blood-corpuscles, and whose volume subsequently increases con-
siderably, is called the serum. If, instead of merely allowing the blood to
stand, it is stirred vigorously with a wooden, or glass, stirring rod, imme-
diately after taking it from the animal, a different coagulum is soon formed
which is known as fibrin. In this case the blood-corpuscles remain for
the most part suspended in the serum. This mixture of serum and blood-
corpuscles is spoken of as defibrinated blood. The difference between the

536 LECTURE XXIII.

two experiments lies merely in the fact that when there is a spontaneous
coagulation of the blood, the fibrin holds the blood-corpuscles within its
meshes and only squeezes out the serum, while by beating the blood the
fibrin is separated from the blood-corpuscles.

The question that interests us first of all is as to the difference between
the plasma and the serum. Whereas the normal blood, as it circulates
through the blood-vessels, consists essentially of only two constituents,
plasma and blood-corpuscles, clotted blood contains three, — namely, the
blood-corpuscles, serum, and fibrin. In both conditions of the blood we
find the corpuscles. They remain unchanged, or at least the red corpuscles
do, as far as we know. The plasma, on the other hand, separates into two
parts, serum and fibrin. The following scheme represents these relations:

Blood

Blood

ood-

L

corpuscles

Fibrin

li

asma
Seru

Blood-clot (when formed
spontaneously)

Blood-corpuscles Plasma

I Serum Fibrin

Defibrinated blood

Fibrin is unquestionably formed from the plasma. It was conceivable
that it is present as such in the blood under normal conditions, being
held in solution while the blood is circulating in the body and only
caused to precipitate under definite conditions. On the other hand, it
was also beheved possible that fibrin is not present as such in the blood,
i.e., the plasma, except in the form of aprehminary stage of its development.
Careful investigation soon showed the latter view to be the correct one. It
is perfectly plain that this peculiar and remarkable phenomenon of the clot-
ting of blood which has attracted the attention of many investigators even
in remote ages, affords to the animal an important means of protection
against undue loss of blood, sufl[icient in most cases to cause the bleeding
to cease.

The first thorough and systematic investigations, upon which our whole
theory of blood coagulation is based, were made by the two scientists
Buchanan '■ and Alexander Schmidt.^ Each of these men, working inde-
pendently, has explained the essential points concerning the clotting of
blood. Buchanan made the important observation that hydrocele fluids

' Proc. Philosoph. Soc. Glasgow, 2, 1844 (1845).

' Arch. Anat. Physiol. 1861, 1862; Pflijger's Arch. 6, 445 (1872); 9, 354 (1874); 11,
291 and 515 (1875); 13, 10.3 (1870). For the literature on the subject, see P. Morawitz:
Ergeb. Physiol. 4, 307 (1905). Other comprehensive works on the clotting of blood to
which we would call attention are, Alexander Schmidt: Die Lehre von den fermenta-
tiven Gerinnungserscheinungen (1S7G); Zur Blutlehre (1892) and Weitere Beitrage
zur Blutlehre (1895); Arthus: Neuere Arbeiten zur Blutgerinnung (1899); E. Schwalbe:
Beitrage zur Cheniie und .Morphologic der Koagulation des Blutes (1900); A. Schitten-
helm: Zentr. Stoffwechs. u. Verdauungs Krankheiten, 6, 143 (1905).

THE BLOOD. 537

which of themselves do not clot, immediately coagulate if a little blood-
serum, or a clot of blood, be added to them. Now blood-serum by itself
does not clot; it is, in fact, formed by the clotting of blood. Thus the
union of two liquids, either of which alone is not capable of forming a clot,
produces coagulation. Buchanan concluded from this, and correctly,
that two substances are necessary for the formation of blood-clot. He
assumed one of these to be fibrin, while the other, probably originating
from the white corpuscles, acted upon the fibrin and converted it into an
insoluble form. Denis ' attempted to isolate this "soluble fibrin." He
first prevented coagulation by collecting the blood in one-sixth its volume
of a saturated sodium sulphate solution. He then allowed the heavier
blood-corpuscles to settle out, and precipitated, by the addition of common
salt, an albuminous substance from the plasma which he had siphoned off.
This substance dissolved in water, but coagulated after a short time; we
will give to it the name of fibrinogen. It may be considered as the ante-
cedent of fibrin. Buchanan recognized the fact that a second substance
was probably necessary to change fibrinogen into fibrin. Our thanks are
due to Alexander Schmidt, however, for showing that the coagulation
process is due to a fermentation. He succeeded in isolating a substance
from the blood-serum which was capable of causing the separation of a
large quantity of fibrin. The substance becomes inactive after it has been
heated to 100° C. Its optimum of activity lies at 37° C. This substance
Schmidt designated as fibrin jcrmenl. By its action upon fibrinogen,
fibrin is formed. The circulating blood does not contain the fibrin fer-
ment. It is formed, according to the experiments of Schmidt, by the
disintegration of the white corpuscles. We must mention here that he
himself did not regard the formation of the fibrin ferment as such a simple
process. He did not assume the presence of an antecedent, but believed
that fibrin was formed from two entirely distinct substances, a fibrino-
genous substance and a fibrinoplastic one. Olof Hammarsten ' dis-
puted this view, and attributed the fermentative action to a conversion of
fibrinogen into fibrin. Other investigation has shown that HammarSten'a
theory is correct.

There is still another important point to mention. .Alexander Schmidt
pointed out that the formation of blood-clot also required the presence of
neutral salts. According to his views, all soluble salts of the alkalies and
alkaline earths reacted similarly. Hammarsten noticed, on the other

' Nouvelles etudes chimiques, physiologiques et m^decines sur les Bubstances albu-
minoids (1856), and M^moire sur le sang (1S59).

' Nova acta Reg. Soc. Scient. Upsala, Ser. 3, 10, 1 (1875); Upsala lakareforenings
forhandlingar, 11, 1876; Pfliiger's Arch. 17, 41.3 (1878); 18, .38 (1878); 19, 563 (1879);
22, 443 (ISSO); 30, 437 (1883). See also Fr^d6ricq: Bull, de I'acad. roy. Belgique, 2
s^rie, 44, 7 (1877).

538 LECTURE XXIII.

hand, that calcium chloride exerts a particularly favorable action upon
the rapidity of the coagulation. The necessity for the presence of lime-
salts was proved clearly by Arthus, and Arthus and Pages.' They showed
that blood collected, as it flows from the animal's body, in a solution
of alkali oxalate does not clot. If, however, a slight excess of lime-
salts is added to this oxalate plasma, a clotting at once takes place.
It is tempting to compare the clotting of blood with the coagulation of
casein by rennin. The latter would correspond to the fibrin ferment.
This ferment changes fibrinogen into fibrin, which may' form an insoluble
calcium salt, and is precipitated. This simple explanation of the clotting
of blood has, however, been shown to be incorrect. The lime-salts must
act in some other way.

In order to understand the clotting of blood, and the processes which
take place in this connection, it is necessary to bear in mind the following
points. In discussing the digestive ferments we were constantly confronted
by the fact that the ferments as such are not given up by the cells, but in
the form of their antecedents, to which in general we gave the name of
zymogens. The transformation of these inactive substances into active
ferments is lirought about by various agents. How was it with trypsino-
gen? We remember that this was changed into trypsin by the so-called
enterokinase which is given up by the epithelial cells of the intestinal
membrane, and is contained in the intestinal juices. Again we remember
that a substance called secretin has been oljtained from the blood, which
incites the gland-cells of the pancreas into greater activity. The secretin
is likewise found in the intestinal membrane in a preliminary stage, which
is activated by acid. We do not know how secretin influences the action
of the pancreas, whether it acts directly upon the gland-cells or indirectly
by increasing the blood-supply. At all events it is evident that the forma-
tion of ferments is a very complicated process, and even when the zymogen
has been formed it does not at all signify that the fermentation will take
place.

In accordance with this aspect, we must next find out whether the fibrin-
ferment, sometimes called thrombin, possesses a zymogen stage in its
development, and if so, how it is brought into activity. Further investi-
gation has in fact shown that the fibrin-ferment does exist originally in an
inactive form. We will call this simply the zymogen of the fibrin-ferment.
This zymogen may be obtained in large amounts from the oxalate plasma
and becomes active only after being treated with calcium chloride solution.
In this way the fibrin-ferment is obtained. This ferment is capable of
causing coagulation in the oxalate plasma, from which the calcium salts

' Arthus: Doctor's Thesis, Paris. 1890. Arthus and Pages: Arch. Physiol. 22, 739
(1890); Arthus: Compt. rend. soc. biol. 45, 435 (1893); Arch. Physiol. 1896, 47, and
Compt. rend. soc. biol. 54, 526 (1902).

THE BLOOD. 539

have been precipitated. According to tliis, it is easy to assume that the
calcium salts effect the activating of the zymogen. The plasma normally
contains only the zymogen of the fibrin-ferment and not the ferment itself.
In clotting blood, the ferment is made active under the influence of calcium
salts, and is now exerting its action upon the fibrinogen. If, on the other
hand, the calcium salts are removed by precipitation with oxalic acid
before the coagulation has taken place, then the blood does not show the
same tendencj^ to form a clot, because the zymogen remains unchanged,
and in this concUtion it is perfectly inactive. Further support for the view
that the calcium salts are only active in this phase of the coagulation, and
that they do not take part directly in the conversion of fibrinogen into
fibrin, is shown by the work of Hammarsten. He pointed out that only
those calcium compounds need be considered which are present in such a
state that the calcium is precipitable by oxalic acid. The oxalate plasma
contains calcium, in addition, which is evidently present in the form of
complex organic compounds. It is possible now to convert fibrinogen
into fibrin by the action of the fibrin-ferment in the absence of lime salts
that can lie thrown down by oxalate. This experiment is perfectly analo-
gous to that with the oxalate plasma. Hammarsten was able also to show
that the fibrin could not be regarded as a calcium compound. We do not
know exactly how the calcium salts cause this conversion of zymogen
into ferment. It is possible that it acts directly upon the zymogen, but
it is likewise conceivable that the calcium salts have an indirect action in
regulating the conditions. For the present, however, we shall consider that
they themselves take part directly in the conversion of the zymogen into
ferment, and that otherwise they have nothing whatever to do with the
formation of the fibrin.

We shall at this place mention that quite recently it has been noticed
that calcium salts exert an activating effect upon trypsinogen. If inactive
pancreatic juice be treated with sodium fluoride it will remain in this con-
dition, and is only activated by the addition of calcium salts. In such a
case as this the calcium salt evidently does not act directly upon the zymo-
gen. If the pancreatic juice be filtered through collodion, then it will no
longer be activated by the addition of the calcium salt. Delezenne,' who
performed this last experiment, believes that some substance is held back
on filtering the pancreatic juice through collodion, which is capable of
activating the trypsinogen. The calcium salts serve in some way to
activate the unknown substance just as it is possible that enterokinase
may have an antecedent. If we apply this observation, which to be sure
has never been explained entirely satisfactorily, to the coagulation of the
blood, then we should have to assume that the zymogen of the fibrin-fer-
ment is activated by a substance which corresponds to enterokinase, and

• Compt. rend. soc. biol. No. 33 (1905).

540 LECTURE XXIII.

that this activator is likewise present in tlie blood in an inactive condition,
and is only changed into the active condition by the presence of calcium
salts.'

We must recall one other fact that we met with in discussing the diges-
tive ferments. We mentioned that the bile, and the intestinal juices in
general, had the property of augmenting the action of the pancreatic juice.
According to our present knowledge, this does not consist merely in chang-
ing zymogen into ferment. The above-mentioned secretions accelerate
directly the fermentation process. Until we understand the nature of
ferments better, there is naturally but little prospect of our being able
to comprehend the accelerating effect. We can only mention the fact,
and state in addition that there are other substances known which
tend to retard the action of ferments without in any way injuring them
directly.

We must also ascertain whether there are substances which tend to
accelerate the action of the fibrin-ferment. It has indeed been long recog-
nized that there are substances which accelerate the clotting of the blood.
Even Buchanan noticed an acceleration produced by certain tissues.
Rauschenbach ^ has proved beyond question that there are substances
present in the cells of the tissues which aid in the formation of the clot. He
found a particularly favorable action on the part of those tissues which
were rich in nuclein substances. Foa and Pellacani ' showed further that
the injection of the juices from various tissues caused intravascular coagu-
lation. This caused a tedious, unfruitful discussion as to whether the
substances present in the cells of the tissues were to be considered as
corresponding to the fibrin-ferment, or whether they merely augmented
its action upon the plasma. The first assumption is a tempting one. In
the first place, according to many observations the leucocytes are the ante-
cedents of the fibrin-ferment. It would be of itself not inconceivable that
other cells in the body should produce similar, or the same, products,
especially as it is well known that after death a coagulation takes place in
the cells which may be considered as perfectly analogous to the clotting of
blood.

Certain observations, however, make it seem more probable that the
coagulation action of the tissue extracts depends upon a quite different
process than the direct introduction of fibrin-ferment. Above all, the work

' It is probably true that the lime salts do not directly cause the activation of the
fibrin-femient zymogen, for it is not easy to see otherwise how the blood can contain
the two substances side by side without their reacting together. Tlie calcium salts are
only brought into activity when the fibrin-ferment zymogen has been changed in some
way.

' Ueber die Wechselwirkungen zwischen Protoplasma und Blutplasma. Dorpat,
1883.

• Arch. Science med. 7, 113 (1883).

THE BLOOD. 541

of Delezenne ' should be mentioned in this connection. He showed that
the blood of birds, reptiles, Batrachia, and fish coagulated but very slowly
of itself. If for example the blood of a bird be carefully removed so that
it does not come in contact at all with the tissues, it can be kept in vessels,
out of contact with dust, for a long time, without forming a clot. By
centrifugalizing such blood, plasma can be obtained which keeps until it
putrefies without the formation of any clot. If, however, a little blood, or
a little juice from the tissues, be added to such cell-free plasma, there is at
once a formation of fibrin. If the blood clots of itself, then we notice that
the clot is formed first at a place where there is present a considerable
amount of leucocytes. This experiment cannot be carried out with
the blood of mammals, for it coagulates too quickly. Evidently their
leucocytes are less resistant than those of the above-mentioned classes
of animals. The objection may be raised that in every case there is
the possibility that active fibrin-ferment may be carried to the blood of
plasma by the juices from the tissues, while the zymogen of this fibrin-
ferment which is contained in the blood itself, for some reason is not
changed into the active form. Morawitz ^ has proved, however, that this
objection is not well founded. He showed that the juice from the tissues,
even in the presence of calcium salts, was incapable of causing a fibrinogen
solution to coagulate. If, however, the extract from the tissues be
added to the blood itself, there is a marked acceleration of the coagulation
provided that the lime-salts are present. In the absence of lime-salts, the
extracts from the tissues are without action.

Alexander Schmidt distinguished between accelerating and retarding
substances for the coagulating of the blood. He believed that these are
present in the leucocytes, and in all other cells of the bodies. The former
class of substances may be extracted with alcohol, while the latter cannot.
We can imagine that the substances which accelerate the clotting of blood
serve to activate the zymogen of the fibrin-ferment. Under normal condi-
tions, i.e., while the blood is circulating through the blood-vessels, there is
an equilibrium between these substances that accelerate and those that
retard its coagulation, whereas when clotting takes place, this equilibrium
is disturbed in favor of the former. Plausible though this hypothesis
may be, it must be emphasized that it is not based upon a careful analysis
of the separate processes.

If we draw a picture of blood-coagulation in accordance with what has
been stated above, we may assume it to be well established that the blood,
or, better, the plasma, contains a substance, fibrinogen, which is an ante-
cedent of fibrin. This transformation of fibrinogen into fibrin is to be

' Compt. rend. soc. biol. 48, 782 ; Corapt. rend. 122, 1281 (1896) ; .\rch. Physiol. 1897,
333; Compt. rend. boc. biol. 49, 462, 489, and 507 (1897).

* Arch. klin. Med. 79, 1 (1904); Hofmeister's Beitr. 4, 381 (1903); 5, 133 (1904).

542 LECTURE XXIII.

regarded as the result of fermentation. The fermentation i.s brought
about by the fibrin-ferment which does not occur as such in the blood but
is probably present in the plasma in an inactive condition, the zymogen of
the fibrin-ferment. In order for the zymogen to become active, a second
substance must act upon it. A true activation must take place. The
e.xact nature of the activator of the zymogen is at present unknown. It
is not impossible that calcium salts bring about this effect. Certain
observations, however, make it appear improbable that the calcium salts
take part directly in the formation of the ferment from its zymogen con-
dition. Some facts indicate that a peculiar kinase is active, which is
itself set free in some way so that it can act upon the zymogen. Appar-
ently it is here that the calcium salts come into play. It may be mentioned,
in addition, that two antecedents of the zymogen have been described in
the literature. One of these, which has not been mentioned, is said to
be activated by acids or alkalies. It has been found, namely, that the
serum from clotted blood, which of itself contains but little active ferment,
will become very active upon the addition of acid or alkali. There is,
nevertheless, no ground for assuming that there are two kinds of zymogen
present. We agree with Morawitz that the fibrin-ferment, for some reason
or other, becomes inactive after the blood-clot has formed. It is simplest
to assume that the fibrin-ferment unites with some other compound,
whereby its active group is rendered inactive. It is equally plausilale that
by means of some intramolecular transformation, perhaps the formation
of an anhydride, the inactive condition of the ferment is again obtained.
The alkali or acid which is added to the serum would in the first instance
set the ferment free, whereas, according to the latter view, the anhydride
form would be lost. At all events, it is unnecessary to assume that the
fibrin-ferment has two antecedents. It is evident from this attempted
explanation, what a complicated process the clotting of blood really is,
and how many different minor processes must be cleared up before we shall
be able to understand the whole phenomenon of blood-coagulation.

The point that is of chief interest to us here, is as regards the nature of
the fermentation. Does a hydrolysis take place, an oxidation, a reduction,
or what? The first assumption only need be discussed. Formerly, it was
believed that the fibrinogen took on water and formed fibrin together with
a substance soluble in water, which was called fibrin-globulin. Recently
every justification for such an assumption has been denied, and mainly
because it is possible to prepare solutions of fibrinogen which will not split
off fibrin-globulin either by clotting or coagulating by heat.' According
to the more recent view, the formation of fibrin results from an intra-
molecular rearrangement of the atoms. There is at present no prospect of
deciding this question definitely. Fibrinogen is an albuminous substance,

' Huiskamp: Z. [)liysiol. Chem. 44, 182 (1905).

THE BLOOD. 543

of which we merel}' know that it is closely related to the globulins, and
fibrin also belongs to the proteins. It is perfectly possible that fibrinogen
consists of several molecules of fibrin which take up water and disintegrate,
whereby eventually fibrin is formed, so that although fibrin is formed from
fibrinogen, the coagulation is essentially a hydrolytic decomposition.
There are no facts known at present which contradict such an assumption.
Under ordinary conditions when the body is wounded, the blood flows
out from the blood-vessels past the tissues. Its clotting is thereby accel-
erated. We must here consider the anomalous behavior of the blood in
hemophilia. This condition is especially characterized by the fact that the
blood does not clot readily. Even from the slightest wounds there is a
tendency to bleed freely. There may be even such a slight tendency
for the blood to clot, that a wound resulting, for example, from the
extraction of a tooth may cause the patient to bleed to death. Hemo-
philia has been of interest to biologists not only on account of this peculiar
condition but also by a further peculiarity which holds almost invariably.
This is the fact that it is hereditary, and, moreover, it is almost always
the male members of the so-called families imth hemophili-c blood, who
inherit this tendency. On the other hand, the female members of the
families alone transmit the anomaly. The following tree shows how this
tendency runs : —

Family I'

I Family II |

<?._ s -<?

f; \ \ I Vr \ \r

• ? ? 9 • 9 •

(47yrs.old) | | |, | 26yrs.old 1 ISyrs.old

I <? (? • 9 <?

9 (19yrs. (loyrs. (12yrs. (4 yrs. (1 yr.

15 yrs. old) old) old) old) old)

old

The symbol <? indicates those male descendants which were normal,
^ indicates those males having the tendency to bleed freely, and 9 rep-
resents the female members of the family. We mention hemophilia at
this place because this anomalous behavior, which is hardly to be regarded
as a disease, inasmuch as the individuals are normal in every other respect,
ought to be of assistance to us in the study of the coagulation of blood.

' Abderhalden: Beitrage pathol. Anat. und allgem. Path. 35, 213 (1903). See
Stahel: Inaug. Dissert. 1903, Zurich, 1880. Hoessli: Inaug. Dissert. Basel, 1885. Sahli:
Z. klin. Med. 56 (1905).

544 LECTURE XXIII.

Here nature has performed for us a physiological experiment. Evidently
some link in the chain of processes from those which start the coagulation,
to those that complete it, is missing. First it was suggested that perhaps
the calcium salts were not present. Direct experiment failed to confirm
this. The behavior cannot result from a deficiency in fibrinogen, for the
blood is found to yield just as much fibrin as normally. The remarkable
thing is the time that it takes for the blood to clot. If a trace of defibrin-
ated normal blood is added to the hemophilic blood, the coagulating is
thereby accelerated.

It is evident that this abnormal blood contains all of the constituents
that are required for forming the clot. It contains sufficient fibrinogen,
sufficient calcium salts, and, as far as we are able to find out, enough of the
zymogen which antecedes the fibrin-ferment; but, on the other hand, one
obtains the impression that an insufficient amount of the agent is present,
which changes the zymogen of the fibrin-ferment into the active state. We
have designated this substance as a kinase, and suggested that it occupies
a similar position to that of enterokinase. Now cases of hemophilia have
been known in which all of the blood did not show this abnormal behavior,
but in which it was restricted to the blood from certain parts of the body,
especially from the blood-vessels in the mucous membrane. While injuries
to the skin, for example, caused a normal bleeding, a very slight injury
to the mucous membrane might cause enormous hemorrhages which could
hardly be checked at all. Now there is no doubt that there is some ana-
tomical anomaly in the walls of the blood-vessels concerned, for mere
scratches which would not ordinarily cause any bleeding at all, now cause
it to take place profusely. But while it is easy to understand that abnor-
mally constructed blood-vessels may be injured more readily, this does not
explain at all why it is that the blood from them does not coagulate.

The characteristic phenomenon with such bleeding is that the blood
constantly oozes through a loose clot exactly as through a sponge. The
coagulum formed is not connected properly with walls of the injured
blood-vessels. At the best, it is merely suspended from walls without
being closely bound to them. It does not fulfill its normal function of
checking the flow of blood. This tends to lead one to suspect that the walls
of the blood-vessels themselves have something to do with the clotting of
the blood, and most probably in the sense that the injured cells give up
something which accelerates the coagulation process. We must designate
this substance as a kinase, and localize its function in the entire process to
that of the preliminary stage; i.e., we must ascribe to it the property of
activating the zymogen of the fibrin-ferment. Consequently a chemical
anomaly is closely connected with this anatomical one. It is not impossible
that further studies based upon these observations will eventually show
what parts of the walls of the vessels take part in the secretion of the kinase.

THE BLOOD. 545

Naturally it does not necessarily follow that all cases of hemophilia result
from the same cause. Perhaps in different cases, different links in the
whole chain of processes may be missing. We have introduced this dis-
cussion here not alone because it apparently sheds light upon the process
of coagulation, a fact to which H. Sahli first called our attention, but
because it seems to us as highly significant that such an extremely localized
anomaly in the whole course of a fermentative action should prove to be
hereditary.

We have up to this point failed to answer the question why the blood
does not coagulate in the blood-vessels of the body under normal con-
ditions. It is a priori not so easy to see why conditions could not pre-
vail within the blood-vessels which would bring the fibrin-ferment into
activity. On the other hand, the fact that the coagulation process depends
upon the harmonious action of several distinct factors, presents several
possibilities whereby the coagulation in the blood-vessels themselves
may be prevented. It is merely necessary to prevent the bringing into
activity of the fibrin-ferment. The following experiment' brings to our
attention still another factor which we have not considered in the coagu-
lation of blood. If blood is collected under vaseline or oil, it remains
perfectly fluid for hours. It can be stirred with a greased rod without its
coagulating. If, on the other hand, the precaution is not taken to grease
the rod, coagulation at once takes place upon stirring. It is even possible
to centrifugalize blood that has been collected in paraffined vessels, and
thus obtain plasma, which similarly will not clot for a considerable length
of time if it is kept in greased or paraffined vessels. On the other hand,
if such blood is poured into an ordinary glass vessel, it will clot immediately.
The contact of the l)lood with something that it can wet, seems to start the
coagulation. As this paraffined plasma, after the calcium salts have been
removed from it, does not coagulate when brought into contact with
foreign bodies, it is out of the question to believe that it contains an active
ferment. By the process of exclusion, we may conclude that the contact
with a foreign body in some waj^ accelerates the transformation of the
zymogen into active ferment. One might assume from this that the reason
the blood does not coagulate in the blood-vessels, with intact intima, is
that the conditions are similar to that of the greased vessels outside of the
body. In fact, we know that if the intima is in any way injured, intra-
vascular coagulation readily sets in, which is called a thrombus when it
merely stops up the blood-vessel which is injured, but is known as embo-
lism when the coagulation influences other blood-vessels as well. The
blood is continually wetting the intima of the blood-vessel walls. The

' Briicke: Virchow's Arch, 12, 100 (18.57). Freund: Wiener Med. Jahrbiicher, 1888,
259, and 1889, 554. Bordet and Gengou: Ann. Institute Pasteur, 17, 822 (1903); 18,
1 (1904).

546 LECTURE XXIII.

fact that coagulation does not take place cannot be due to an insufficient
adhesion. The uninjured walls of the blood-vessels appear to exert a
restraining influence upon the coagulation. It is indeed possible that
they also produce a secretion of a negative catalytic nature.

We have mentioned that it is possible to prevent the clotting of blood
outside of the body by collecting it in a solution of ammonium oxalate.
Sodium fluoride acts similarly. The cause of the failure of coagulation to
begin is to be considered as due to the precipitation of the lime-salts
as oxalate or fluoride. The addition of neutral salts in sufficient concen-
tration, or cooling the blood, also tends to prevent the formation of a clot.
The influence of both measures is due to a restraining of the action of the
ferment. Now we know of a number of substances which may be intro-
duced into the circulation so that the blood which is subsequently removed
will not coagulate. Commercial peptone is such a substance.' We may state
in this connection that peptone acts differently in the case of different
animal species, and in fact with one individual of a species it may prevent
coagulation, in another merely retard the formation of coagulum, while
in a third member of the same species it will be apparently without effect.
This indicates that the influence of peptone upon the process of blood-coagu-
lation is not so simple as is the case with oxalic acid for example. It is
important also to find that it takes from ten to fifteen times as large a dose
of peptone to prevent the coagulation in a test-tube as is required in the case
of injection into the organism. It has never been found possible to localize
in any way this action of the peptone, although apparently, in the light of
recent investigations, the action is not due to the peptone itself, but to
impurities present in commercial peptone. How this effect is produced
is entirely unknown to us. It has been suggested that it causes the for-
mation in the body of substances which tend to prevent the coagulation.
It has been established that peptonized blood contains all the elements
which are considered as necessary for the clotting. In spite of this fact
the harmonious course of the entire chain of processes is in some way
disturbed. We can indeed imagine that by the failure of some one
of the substances which aid in the coagulating process, the clotting is
prevented. The disturbance is at all events to be sought in the group of
processes by which the activating of the zymogen of the fibrin-ferment is
effected.

Other substances are known which act similarly to peptone. We will
mention the serum of Mura-na ' and the extract of crabs' muscles and of

' Schmidt-Mulheim: Arch. Anat. Physiol. 180, 3.3. Albertoni: Zentr. med.
Wissensch. 1880, No. 32. Fano: Arch. Anat. Physiol. 1881, 277.

2 Mosso: Ann. chim. farm. 8, 198 (1S88), and .\rch. exper. Path. Pharm. 25, 111
(1891). Delezenne: Arch, physiol. 646 (1897), and Corapt. rend. soc. biol. 49, 42 and
228 (1897). Heidenhain: Pfliiger's Arch. 49, 209.

THE BLOOD. 547

snails. The characteristic of these substances which prevent clotting is,
to repeat, that they evidently do not themselves directly affect the coagu-
lation process, but excite the organism to the formation of products which
tend to prevent the blood from forming a clot.

Besides these substances which exert a secondary effect upon the
coagulation, we know of substances which directly prevent it. To these
substances belongs hirudin, which, by reason of the extensive studies of
Jakobj, has recently excited much interest. Hirudin is formed in the oral
glands of leeches.' It is quite stable towards heat and is soluble in water.
After injection into the organism it appears unchanged in the urine. It
is not yet perfectl}^ clear how hirudin acts. It is apparently able to neu-
tralize a part of the fibrin ferment.^ It is still an open question how we
can best picture this process. It is usually assumed that the active ferment
possesses groups which enable it to react with definite groups of other
compounds. These groups impart to the ferment its specific nature. If
now the ferment comes in contact with a substance which is capable of
engaging these groups, i.e., combining with them for example, the ferment
then becomes inactive. Substances like hirudin have been obtained from
other blood-sucking animals, e.g., from the wood-tick (Ixodes ricinus) and
from Anchylostomum caninum.

It is an old observation that the blood of animals which have died from
snake-bite, often does not coagulate.^ The poison from the cobra especially
has been carefully studied. It is supposed to contain a substance which
acts upon the kinase, i.e., the activator of the zymogen of the fibrin-ferment.
It is clear that if the function of this activator is disturbed, a coagulation
cannot take place.

Besides the substances which prevent the coagulation of blood, we know
of others which accelerate it. In this respect we will recall the effect of
calcium salts. Their internal application is said to favor the formation of
blood-clot. Another substance which is used much more extensively
for this purpose is gelatin.'' It is altogether impossible to state why this
property should be ascribed to gelatin, and if it really does exert the
desired effect it is still more difficult to explain it. At all events, the state-
ments concerning it that are to be found in the literature are very con-
tradictory.

We have up to the present time intentionally disregarded a question of

' Haycraft: Arch. e.xper. Path. Pharm. 18, 209 (1884). Franz: ibid. 49, 342 (1901).
Andreas Bodong: ibid. 52, 242 (1904).

2 Fuld and Spiro: Hofmeister's Beitr. 5, 171 (1904). P. Morawitz: Arch. klin. Med.
79, 4.32 (1904).

' Fontana : On Poisons. London, 1787. Morawitz: Arch. klin. med. 80, 340
(1905).

' Dastre and Floresco: Compt. rend. soc. biol. 48, 243 and 358; Arch, physiol. 28,
302.

548 LECTURE XXIII.

deep significance, namely, that of the origin of fibrinogen. This is something
of which we have no positive information. Apparently the liver plays an
important part in its production. P. Nolf ' found that after extirpation
of the liver the fibrinogen content of the blood decreased rapidly. Experi-
ments performed by M. Doyon, A. Morel, and N. Kareff - point in the same
direction. They showed that after sub-acute poisoning of dogs with phos-
phorus oil, which causes a fatty degeneration of the liver, there is a decrease
in the fibrinogen content of the blood plasma, which lessens the coagula-
tion power of the blood. With a cock it was not found possible to cause
a fatty degeneration of the liver by phosphorus poisoning, and it was
likewise impossible in this case to cause fibrinogen to disappear from the
plasma. It is entirely impossible to draw binding conclusions from these
experiments, for both the extirpation of the liver and poisoning by
phosphorus are attacks whose effect upon the action of the whole
organism cannot be disregarded. It is possible that the liver not only
influences the production of fibrinogen, but other phases of the coagulation
process as well. We shall expect further experiments in this direction.

In the coagulation process we have become acquainted with a very
essential propertii of the blood. We shall now turn to the individual con-
stituents of defibrinated blood, the serum and blood-corpuscles. The
former consists chiefly of two different albuminous substances, a globulin
and an albumin. We shall not stop here to discuss the unedifying question
as to whether these proteins are simple substances or not. This cannot be
decided in the light of our present knowledge, and even if it is possible by
fractional precipitation, or by "salting out," to effect a separation into
simpler constituents, but little gain is made in our knowledge of these two
proteins, for even these fractions cannot be characterized, by the means
now at hand, nor upon the present basis of protein chemistry, as simple
substances. Besides these proteins, we find varying amounts of fat in
blood-serum. After a meal rich in fat, the amount present in the serum
may become so large as to give it a milky appearance. Serum invariably
contains cholesterol and lecithin, and in fact the former is, as we have
already stated, largely present in the form of fatty-acid esters. A sugar,
rf-glucose, is also present in serum. The amount of the latter varies, but
only within narrow limits.

We have already seen that the blood, besides providing nourishment,
also serves to carry the end-products of metabolism away from the cells.
For this reason we constantly meet with such products in the blood. It
is certain that they belong for the most part to the plasma, or its serum.
Their presence remained for a long time undiscovered, because from
moment to moment but small amounts of such substances are present.

' Bull. Acad. roy. Belgique, 1905, 81.
' Compt. rend. 140, 800 (1905).

THE BLOOD. 549

As soon as they are formed, they are given up by the cells to the blood
and immediately leave the bod}*. Such substances are urea, uric acid,
creatine, hippuric acid, and conjugated glucuronic acids, all of which we
may in a sense regard as end-products of metabolism. Blood-serum is
never perfectly colorless. It always has a yellow tint, the color being
ascriljed to a certain dyestuff, called lutein. Its chemical nature is
wholly unexplained. Serum always contains inorganic constituents, and
the amount appears to be very constant. It would be highly interesting
to have definite knowledge concerning the distribution of the inorganic
substances, and above all concerning the way they are combined in the
blood, plasma, and serum. Unfortunately, there are no known methods
for giving us such information. At present we are forced to rely upon the
chemical examination of the ash, the results of which naturally have but
a relative value. In this way we are able to ascertain what constituents
are present in the ash, but we obtain absolutely no information as to how
the phosphoric acid, for example, is combined in the blood or plasma.
This phosphoric acid may arise from inorganic phosphates, or from Organic
phosphorus compounds, such as lecithin, nucleic acid, etc. The value of
an ash analysis can be increased bj' attempting to determine in what dif-
ferent way the respective amounts of the constituents may have been
combined. It would be, of course, likewise desirable to obtain bj' physico-
chemical methods some idea as to the content of the blood and of the
plasma in electroh'tes and non-electrol_ytes. As the most important
result of physico-chemical investigation of the blood, we will mention the
highlj^ interesting observations of Hoeber ' that the concentration of the
hydroxyl ions in blood-serum and in the blood is almost exactlj' the same
as that of distilled water. Both liquids are from this point of view to be
considered as neutral.

Blood always contains cells, namely, the red and white corpuscles.
Whereas the latter are to be regarded as true cells, the former are, in man
and mammals, not to be considered as perfect cell-structures. Only in
the beginning of their development do they possess a nucleus, which they
lose as soon as they become active in the blood. The red corpuscles of
birds, reptiles, amphibia, and fishes do, however, contain nuclei. In spite
of extensive investigations but little is known concerning the chemical
construction of the red corpuscles. It is true that we know fairly well
what components are present, but we do not understand how they are
combined. The red corpuscles do not possess any true membrane. It
has been assumed that they consist of stroma filled with liquid.^ They are

' Pfluger's Arch. 81, 522 (1900). G^za Farkas: Mathematikai ^s term^szettudo-
mdnyi ^rtesfto, 21, Vol. 1 (1902). P. Fraenkel: Pfluger's Arch. 96, 601 (190.3).

' H. J. Hamburger: Osmotischer Dnick und lonenlehre in den raedizinischen Wis-
senschaften, Wiesbaden, 1902. Cf. Rollett; Pfluger's Arch. 82, 199 (1900).

550 LECTURE XXIII.

enveloped with a substance similar to fat which forms a semi-permeable
wall. It is certain that the red blood-corpuscles, also called erythrocytes,
do not take up all substances. The wall will not allow many salts to pass
through, though it is easily penetrated by water. If the erythrocytes are
placed in a solution of common salt, whose osmotic pre.ssure corresponds
exactly to that of blood-plasma, the blood-corpuscles will remain unchanged.
Such a salt solution is said to be isotonic. Its concentration is different
for different species of animals, and is called a "physiological salt solution."
In the case of mammals such a solution contains 0.9 per cent of sodium
chloride. If there is more salt present in the solution, it is said to be
hyper isotonic, in which case the red corpuscles will give up water to
the solution and shrink in size. Conversely in a hypisotonic salt solution
(one containing less salt than an isotonic solution) the corpuscles take
up water and swell. This swelling may take place to such an extent
that the red corpuscles lose the characteristic pigment which passes
into solution. In this case the blood undergoes a peculiar transfor-
mation. Whereas it was opaque before, it now becomes a clear, trans-
parent, red-colored liquid. The blood is said to be "laked." ' In the
laked blood, the blood-corpuscles robbed of their hemoglobin, the so-called
"shades," are found in which only stroma is present. The "shades"
appear under the microscope as colorless structures, often retaining the
form of the erythrocytes. The red corpuscles are not impenetrable to all
substances. Urea, for example, is taken up by the blood-disks. If urea
is added to blood it distributes itself equally between the blood-corpuscles
and the plasma. Its solutions, therefore, exert no osmotic pressure upon
the red corpuscles. The latter behave in urea solutions of all concentra-
tions exactly as in distilled water. They give up their hemoglobin to the
urea solution ; this is not the case if the urea is added to an isotonic solution
of common salt. We know of certain substances, such as ammonium
chloride, for example, which behave differently from urea in the last case.
The wall surrounding the red corpuscles is readily penetrated by this salt,
and the hemoglobin goes into solution even if the ammonium chloride is
added to an isotonic common salt solution. Ammonium chloride, there-
fore, has a poisonous action upon the blood-corpuscles. A great many
experiments have been carried out with regard to the permeability of the
red corpuscles. .A.t present they do not give us much information con-
cerning the behavior of the blood-corpuscles in the blood itself, and con-
cerning the substances dissolved in the plasma. We are not justified in
applying experiments performed under peculiar conditions to the blood
itself as it exists in the living organism.

The exit of the pigment from the red corpuscles, a process which is

' Hans Koeppe: Pfluger's Arch. 103, 140 (1904); ibid. 107, 86, 183 (1905).

THE BLOOD. 551

designated as hemolysis, can be brought about in quite a number of different
ways, as, for example, by freezing tiie blood and then thawing it. Hemo-
lysis is also effected by certain bacterial metabolic products, by those of the
higher plants, and also those of animals. We shall subsequently take up
this process more in detail.

The constituent of the red corpuscle which has been best studied
as regards its functions, is the pigment of blood, hemoglobin, which we
have already met with in the discussion of the respiratory exchange.
Before discussing its chemical construction, we will consider the above-
mentioned, cellular constituent of the blood, the white corpuscles, and
briefly take up the composition of the blood as a whole, and its content of
individual substances. The white corpuscles are fully endowed cells.
They are not uniform, but occur in various shapes and sizes. It is extremely
difficult to say much about the sphere of activity of these bodies, also
called leucocytes. Their function has never been satisfactorily explained.
They have frequently been designated as agents of transportation. It is
very probable that they play an important part in this function of metabo-
lism, and accomplish the exchange of substance between the cells of
different organs. The number of leucocytes can increase extraordinarily
under certain conditions. This phenomenon is most strikingly illustrated
in cases of infection, in which case the seat of infection is, under normal
conditions, surrounded very quickly by a cordon of leucocytes. They
are by no means limited to the blood circulation. They can leave this
and penetrate into the tissues. The white corpuscles play a quite different
part in the blood from that of the red ones. They are not pecuhar to the
blood, but merely make use of it as a vehicle. They enter and leave it
quite at will. They are to be considered as independent entities. This
is evident from the fact that they are independent of the nervous sys-
tem and can move themselves forward, autonomously like the amoebee,
by sending out pseudopodia. It is possible that the blood contains,
besides leucocytes, which are only temporarily present, others which
stand in more intimate relations to the blood. We are not at all sure
whether we are to regard the white corpuscles as forming a physiological
unit, or whether certain of them have special tasks to fulfill. Our experi-
ence with pathological processes makes it seem more probable that differ-
ent tasks fall to leucocytes of different forms. The large accumulation
of leucocytes during intestinal digestion remains absolutely unexplained.
It is extremely probable that they in some way take part in the digestive
process, being perhaps active in the assimilation of the food. The fact
that they are able to take up substances directly, and transport them away,
is shown, for example, by observations concerning the absorption of iron.
It is not at all difficult, particularly after administration of inorganic
iron salts, to find, by testing with ammonium sulphide, many white

552 LECTURE XXIII.

corpuscles heavily laden with particles of iron, which they carry to the
nearest lymphatic. Many discoveries show distinctly that the leucocytes
endeavor to carry away foreign substances from the body. It may be
regarded as positively established that they play an active part during
infectious diseases in striving to make the injurious products of the metabo-
lism of micro-organisms harmless, although it is going a little too far to
assume that the leucocytes alone have this tendency. The leucocytes
also serve to disintegrate dead tissue. In this direction, the solution of
the masses of fibrin, which fill the bronchi and the finest bronchioles during
pneumonia, is very interesting. There is in this case, as we have already
indicated at another place, a regular digestive process that comes into
play. The fibrin is decomposed into its constituents and these are
resorbed.

We cannot say much concerning the structure of the white blood-
corpuscles. They contain, as cells, all those constituents which we usually
meet with in cells. There is not much use in mentioning these constituents,
for we are not able at present to draw any conclusions from them, or from
their union with the other building stones of the protoplasm and nucleus,
concerning the participation of this or that substance in the exercise of
definite functions. As soon as our investigations reach the cell, the
enigma is too great.

In addition to the leucocytes we find the blood-plates, which we have
already mentioned. The'se are colorless, gummy disks of a round form.
They are said to possess all the characteristics of true cells, and to be
also capable of active, amoeboid movement. They undoubtedly partici-
pate in the clotting of blood. It is, however, still a disputed question as
to the point in the entire coagulation process at which their activity
begins.

Let us now return to the composition of the blood itself. We must at
once state that .the blood, in its natural state, is almost never utilized for
quantitative analytical determinations. Almost all of the investigations in
this direction have been made with defibrinated blood. In the first place, we
are interested to know the relative amounts of blood-corpuscles and serum.
This varies with different kinds of animals, and even in different animals
of one and the same species. Moreover, the estimation of the number of
blood-corpuscles, and the amount of the serum, cannot be made very
exactly. It is an indirect determination. We will briefly mention here
that method upon which the figures that we shall give below have been
based. It is that of Hoppe-Seyler.' The blood-corpuscles may be
separated from the serum by means of the centrifuge. By repeatedly
stirring the blood with an isotonic salt solution, and renewed centri-

' Handbuch der physiol. und pathol. chem. Analyse, p. 272 (1883).

THE BLOOD. 553

fugalizing, the serum lying between the separate blood-corpuscles is
eventually removed. In these blood-corpuscles we can determine the
sum of the hemoglobin and protein. If in addition the hemoglobin and
albumin content of the total blood and the albumin content of the serum
are determined, then from these values the relative amounts of serum and
blood-corpuscles in the blood as a whole can be estimated. We may cite
an example:'

One thousand grams of defibrinated beef-blood contain on an average
172.9 grams of hemoglobin plus albumin.

In the blood-corpuscles from 1000 grams of the same blood there were
found 124.0 grams of hemoglobin plus albumin. 1000 grams serum con-
tained 72.5 grams of albumin.

In the serum from 1000 grams of blood there was present, therefore,
172.9 - 124.0 = 48.9 grams of albumin.

Accordingly, we may compute the amount of serum in 1000 grams of
defibrinated blood as follows:

48 9

~x-? 100 = 67.45 per cent serum.

100 — 67.45 = 32.55 per cent blood-corpuscles.

After having established the relative amounts of serum and blood-
corpuscles, then from an analysis of the blood as a whole, and of the serum
alone, we can estimate how much of each substance is present in the blood-
corpuscles.

G. von Bunge ' has shown, by means of the following observation, that
this method gives us, within certain limits, quite reliable results. The
blood-corpuscles of pig's blood contain no soda. This enables us to com-
pute the relative amounts of serum and blood corpuscles by merely deter-
mining the amount of soda in the blood, as a whole, and in the serum by
itself.
Bunge found in 1000 grams of defibrinated pig's blood, 2.406 grams Na20

in " " " the serum 4.272 grams Na20

Pig's blood therefore contains

.,,„,, . 100 = 56. .3 per cent serum.
4.2/2 '

100 - 56.3 = 43.7 per cent blood-corpuscles.

Now if the calculation was made with reference to the albumin content,
as in the first case cited, he obtained the values: 56.6 per cent serum and-
43.4 per cent blood-corpuscles.

' Cf. Abderhalden: Z. physiol. Chem. 23, 521 (1897); 25, 67 (1898).
' Z. Biol. 12, 191 (1876).

554

LECTURE XXIII.

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THE BLOOD.

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556 LECTURE XXIII.

The results obtained in the analysis of the blood of different species of
animals are given on pages 554 and 555/ now emphazing, however, that
the analyses of the ash have only a relative value, but on the other hand
such values may well serve, and in fact have served, as a foundation for
further inquiry, although the methods employed are not yet such that the
analytical results will prove fruitful in all directions.

From the table it is evident that the serum from various animal species
is of much the same composition. There are, however, marked differences
in the composition of the blood as a whole, and of the blood-corpuscles.
It is interesting that the blood of related animals is very similar. This
is apparent when we compare, for example, the relative amounts of the
separate constituents of the blood-corpuscles of the carnivora with those
of the ruminants. It is certainly not without significance that the blood
of both these families contains considerable soda, while that of the horse,
pig, and rabbit contains none at all. Certain constituents, as, for example,
sugar, fat, and lime, are apparently wanting in the blood-corpuscles. It
is rather questionable whether we can assume that the substances are
entirely absent. The methods employed are not sensitive enough to make
such a decision possible. To be sure, the fact that this result has been
repeatedly obtained speaks in favor of its correctness. We may call
attention to the fact that quite recently glucuronic acid has also been
found in the blood-corpuscles.

We should mention the fact that the amount of blood contained in dif-
ferent animals has been estimated. A sample of blood was taken from the
animal in question, after which it was bled to death, and the blood-vessels
washed out with water, until the latter came out perfectly clear and color-
less. The wash-water was mixed with the blood, collected, leaving out
the first sample, and the total volume estimated. Then the first sample
was diluted with water until it corresponded in shade with the other
mixture. In this way it was easy to compute the amount of blood con-
tained in the animal.

This method is not very accurate, and there are several sources of error.
It is, in fact, impossible to remove all the blood from the body in such a
way. In the case of dogs the weight of the blood amounts to from seven
to nine per cent of the dog's weight, in rabbits the blood corresponds to
from five to nine per cent, while in man it is only from one-sixteenth to
one-thirtieth of the body-weight.

In human beings, a cubic millimeter of blood contains on an average
5,000,000 red corpuscles in the case of males, and 4,500,000 in females.
There is usually one white corpuscle for every 350 to 500 red ones. Nat-
urally these values vary according to the blood-vessel from which the

' E. Abderhalden: Z, physiol. Chem. 25, 67 (1898).

THE BLOOD. 557

sample is taken. We know, furthermore, that certain conditions greatly
affect these values. Thus it is well known that when a large amount
of water is passed, the blood may become thickened, while inanition has
the same effect. Vasomotor influences also can cause changes in the
composition of the blood throughout the entire organism. More and more
it has become recognized that it is not always possible to draw conclusions
concerning the behavior of the blood as a whole from the examination of
a single sample. For experimental work it is naturally most satisfactory
to withdraw all of the blood, but when this is not possible, it is always
advisable to make several examinations of different samples, remembering
at the same time that we are dealing with only relative values.

LECTURE XXIV.
BLOOD AND LYMPH.

In discussing the respiratory exchange, we called attention to the
important part played by the red blood-corpuscles in this process; namely,
their significance in the transportation of oxygen. We mentioned the
important fact that it is not the entire red corpuscle which combines with
the oxygen, but that this power is limited to the pigment contained in it,
which is known as hemoglobin. This substance is not a simple compound.
It consists of two components, which, according to their chemical nature,
belong to two entirely distinct classes. One of these components, globin,
is a protein. On account of the relatively large proportion of bases which
it contains, and especially of histidine, it is classed with the histones. We
have already mentioned that this classification is to be regarded as a
temporary one. Globin contains the same constituents as are usually
present in proteins.' The other component, which may be separated
fairly easily from the globin, contains iron and is designated as hemochro-
mogen. In the presence of oxygen the latter is readily oxidized to hematin.
In spite of a great deal of careful investigation, our knowledge concerning
the nature of the combination between globin and hemochromogen is still
very incomplete. We merely know that about 4 per cent of hemochromogen
can be obtained from hemoglobin.^ It is still unsettled whether we are
justified in assuming that one molecule of globin unites with one molecule
of the iron-containing constituent, or whether several molecules of globin
are in combination with a single molecule of hemochromogen. There is
in fact no absolute proof at hand that even globin itself is a simple
substance. We are emphasizing these uncertainties, which in part
have been mentioned elsewhere, because hemoglobin has been usually
chosen as a foundation for the calculation of the molecular weights of
proteins.

When oxygen combines with hemoglobin, oxyhemoglobin is formed, and
this compound crystallizes readily. From squirrels it crystallizes in six-
sided plates of the hexagonal system, while that from other animal species
crystallizes in needles, prisms, tetrahedrons or plates of the orthorhombic
system. The solubility of the oxyhemoglobins from different species of
animals is widely different. That from dogs, for example, is less soluble

' Abderhalden: Z. physiol. Chem. 37, 484 (1903).
' F. N. Schulz: ibid. 24, 449 (1898).

558

BLOOD AND LYMPH.

559

than that from cats.' More soluble, and for that reason more difficult to
prepare, are the oxyhemoglobins from the l)lood of men, cattle, and pigs.^
It has been attempted to draw conclusions concerning the uniformity or
differences in the different kinds of oxyhemoglobin by stud3dng their ele-
mentary compositions. We shall cite a few of such analyses in the table
below, but will state again, that the elementary composition of such com-
plicated compounds signifies scarcely anything at all. Even if it were
possible to decompose hemoglobin quantitatively into simpler components,
we would not be justified in assuming, if we obtained the same relative
amounts of the various constituents, that the different kinds of hemoglobin
were uniform. It may be that the various amino acids are arranged in
a different order in the globin molecule, to say nothing of the various possi-
bilities for the formation of isomers. We hold that it is extremely essential
to emphasize the fact that the elementary analyses of proteins and their
complicated cleavage-products should only be used with great caution as
a basis for drawing conclusions, or for further investigations, and that the
real value of each ultimate analysis is but very slight.

ELEMENTARY ANALYSIS OF OXYHEMOGLOBIN.

In per cents.

Horse's blood . .
Dog's blood . . .
Cat's blood . . .
Pig's blood . . .
Beef blood ....
Guinea pig's blood
Squirrel's blood .
Goose's blood . .
Hen's blood . . .

54.75 6.98

54.57 7.22

54.60 7.25

54.17 7.38

54.42' 7.18

54.12 7.36

54.09 7.39

54.26 7.10

52.47 7.19

N

S

0

Fe

17.35

0.42

20.12

0.38

16.38, 0.57

20.43

0.34

16.52! 0.62

20.66

0.35

16.23! 0.66

21.37

0.43

17.45' 0.48

20.07

0.40

16.78! 0.58

20.68

0.48'

16.09' 0.59

21.44

0.4'

16.21' 0.54

20.69

0.43

16.45

0.86

22.5

0.34

0.34'
0.20*

We find from these analyses that the hemoglobin of mammals contains
the elements carbon, hydrogen, nitrogen, sulphur, oxygen, and iron, while
that of birds contains phosphorus in addition. It is very questionable
whether the phosphorus content is due to a peculiarity of the oxyhemo-
globin in birds or whether it is not rather due to an impurity. We remem-

' Abderhalden: Z. physiol. Chem. 24, 545 (189S), and F. Kriiger: Z. BioL 26, 469
(1890), and Z. physiol. Chem. 25, 256 (1898).

' G. Hiifner: Beitrage zur Lehre vom Blutfarbstoff (1887).

' Abderhalden: Z. physiol. Chem. 37, 484 (1903).

* A. Jaquet: Dissert. Basel, 1899, and Z. physiol. Chem. 12, 285 (1888).

5 J. C. Otto: iUd. 7, 57 (1882).

° According to the author's analyses.

' Hoppe-Seyler: Med.-Chem. LTntersuchungen, p. 366 (1868).

560 LECTURE XXIV.

ber that the red blood-corpuscles of birds contain nuclei and considerable
amounts of nuclein substances. It is perfectly possible that the presence of
such an impurity accounts for the apparent phosphorus content in the hemo-
globin of different species of birds, whose blood has been studied. This
assumption appears more probable when we state that the oxyhemoglobin
from birds has never been prepared in a satisfactory manner, nor purified
to the extent accomplished with that from animals, and, moreover, if we
examine the beautifully formed crystals under the microscope, we shall
find that they may include within themselves considerable amounts of
impurity. In the hemoglobin from horses, pigs, and cattle, two atoms of
sulphur are present for each atom of iron, while in the blood of dogs, the
iron is to the sulphur as 1 : 3. We may also mention the fact that the
various oxyhemoglobins contain different amounts of water of crystalliza-
tion. It is still an open question whether the oxyhemoglobin from one and
the same species of animals is always identical. C. Bohr '■ holds that this
is not the case. He believes he has proved that differences exist by deter-
mining the power of combining with oxygen in different fractions of crystals
from a single kind of blood. Hufner,^ whose investigations in this field
have been thorough and most carefully made, holds that such an assumption
is not justifiable. It must be admitted that it is not easy to prove beyond
all doubt that there is an actual difference in different o.xyhemoglobins.
There is always the possibiUty that the observed differences may arise from
secondary changes which have taken place in the oxyhemoglobin that is
under examination.

As regards the combination of the oxygen in hemoglobin, we have
already seen that only the hemochromogen takes part in this, and that the
iron is of much significance here.

The spectroscopic behavior of oxyhemoglobin is very characteristic. A
dilute solution shows in the spectroscope two absorption bands in the yellow
and green, between the Fraunhofer lines D and E. The band near the D
line is narrower than that near the E line. Arterial blood gives the same
absorption spectrum on account of the presence of oxyhemoglobin in it.
We must also add that reduced oxyhemoglobin, the true hemoglobin, like-
wise shows characteristic absorption bands. A solution of hemoglobin,
of not too great a concentration, shows a single broad band, not very
sharply defined, lying between the D and E lines; in fact, this band extends
a little beyond the D line into the red end of the spectrum. Venous blood
shows such a spectrum, although, except in cases of suffocation, there is
always some oxyhemoglobin present. The greater part of the o.xyhemo-
globin in such blood has, however, been reduced. Consequently venous
blood does not have the bright red color of arterial blood. It is darker

' Zentr. Physiol. 4, 249 (1S90).
' Arch. Anat. Physiol. 1894, 1.3Q

BLOOD AND LYMPH. 561

and has a more violet color. The shade of color naturally varies accord-
ing to the ratio of the oxyhemoglobin to the hemoglobin. Hemoglobin
is more readily soluble in water, and for this reason more difficult to prepare
and maintain in a crystalline form. It may be obtained easily from oxy-
hemoglobin by the withdrawal of oxygen, and this may be accomplished
by placing the oxyhemoglobin in vacuum, conducting an indifferent gas
through its solution, or by the use of a reducing agent. Beautiful crystals
of hemoglobin are also obtained by allowing a solution of oxyhemoglobin
to stand for some time in a sealed glass tube.' The oxygen of the oxy-
hemoglobin is gradually consumed, and hemoglobin is formed by the
reduction.

Carbon monoxide ^ may take the place of oxygen in the oxyhemoglobin
molecule. It evidently is fastened at the same part of the molecule as the
oxygen, for it replaces the latter by its action upon oxyhemoglobin, and
makes it incapable of combining with oxygen except in the presence of a
large excess of the latter gas. It is herein that the poisonous action of
carbon monoxide lies. Carbon-monoxide-hemoglobin can be obtained in a
crystalline form, its crystals being isomorphous with those of oxyhemo-
globin. Its absorption spectrum is very similar. It also shows two absorp-
tion bands, which are, however, nearer the violet end of the spectrum. The
action of reducing agents does not have the effect upon its spectrum that
is obtained with oxyhemoglobin. The two absorption bands do not dis-
solve into one band, or at least not within a short time.

Hemoglobin is also capable of combining with nitric oxide,' NO, and
this last gas is even capable of driving carbon monoxide out of its combina-
tion in the blood. Nitric-oxide-hemoglobin is also crystalline and very
stable. It shows an absorption spectrum very similar to that of oxy-
hemoglobin except that the bands are paler. It is even less affected by
reducing agents than is the carbon-monoxide compound.

The action of hydrogen sulphide * upon oxyhemoglobin gives rise first
of all to the formation of hemoglobin. Then, little by little, a greenish-
black coloration is formed, which reminds one of the appearance of a trace
of ferrous sulphide. This green shade is due to the formation of sulph-
hemoglobin, which, however, has never been prepared in a pure state. It
may be distinguished from hemoglobin by means of its spectral behavior.

' G. Hiifner: Z. physiol. 4, 382 (1880). If oxyhemoglobin is allowed to stand for
two weeks in a quiet place at 40° C, crystals from 1 to 2 centimeters long can be
obtained.

' G. Hiifner: Arch. Anat. Physiol. 1895, 209, 213. Hiifner and Kiister: ibid. 1904,
387.

' L. Hermann: Arch. Anat. Physiol. 1865, 409. Hufner and Reinbold: 1904,
Suppl. II, 391.

* Hoppe-Seyler: Zentr. Med. Wissensch. 1863, No. 28, p. 433. T. Araki: Z.
physiol. chem. 14, 405 (1890). E. Harnack: Ibid. 26, 558, (1898-99).

562 LECTURE XXIV.

It shows one absorption band in the green, and another in the orange-red,
between the C and D Hnes. If hydrogen sulphide acts upon hemoglobin
in the presence of oxygen, the hemoglobin is completely decomposed, so
that the product formed, as well as its derivatives, no longer shows a char-
acteristic absorption spectrum.

Quite different from the above compounds is carbohemoglobin, in which
carbon cUoxide is present, but combined at a different place in the molecule
from that occupied by the oxygen in oxyhemoglobin. In fact, carbon
dioxide and oxygen are taken up by hemoglobin quite independently of
one another. The carbonic acid is evidently combined with globin, while
oxygen combines with hemochromogen.

Methemoglobin ' also occupies a unique position. It corresponds to
oxyhemoglobin in its composition, and differs from it merely in holding
the oxygen in a firmer state of combination. It may be formed from the
latter on standing, or be prepared by the action of various agents, such
as iodine, chlorates, permanganates, nitrites, nitrates, palladium hydride,
pyrogallol, quinol, or ozone. The formation of methemoglobin has also
been observed by the action of aniline, toluidine, acetanilide, acetopheneti-
dine, and glycerol upon oxyhemoglobin. Methemoglobin may be formed
even in the circulating blood, when it comes in contact with substances
such as amyl nitrite, nitrobenzene and antifebrin.

The oxygen cannot be removed from methemoglobin by reducing the
partial pressure of this gas. At present it is not known just how this
transformation of oxyhemoglobin into methemoglobin is effected. It
has been established positively that both compounds contain the same
amounts of oxygen. Reducing agents tend to convert methemoglobin
back into oxyhemoglobin. Methemoglobin crystallizes in brownish-red
needles, prisms, and also in six-sided plates. It is most readily prepared
by adding potassium ferricyanide solution to a solution of oxyhemoglobin
and then, after cooling to 0° C, adding one-fourth its volume of alcohol.
In acid solutions methemoglobin shows an absorption spectrum of a band
in the orange-red between the C and D lines, and a second paler band in
the blue between the G and F lines. Besides these absorption bands,
the acid solutions show two other bands in the same place as the bands
which characterize the spectrum of oxyhemoglobin. It seems probable
that these last two bands are not characteristic of methemoglobin, but
are due to the presence of some oxyhemoglobin as impurity. In alkaline
solutions methemoglobin shows three lines, one on either side of the D line,
and one near the E line. The spectrum of methemoglobins is, in fact,
very similar to that of hematin.

' Hoppe-Seyler: Zentr. med. Wissensch. 1863, No. 28. G. Hiifner: Z. physiol.
Chem. 8, 366 (1884). Hiifner and Otto: ibid. 7, 65 (1882-83). A. Jiiderholm: Z.
Biol. 16, 1 (1880); 20, 419 (1884). R. von Zeyneck: Arch. Anat. Physiol. 1899, 460.

BLOOD AND LYMPH. 563

It is highly probable that hemoglobin not only occurs in the red blood-
corpuscles, but in the muscles as well. The latter likewise contain a red
pigment which cannot be washed out by way of the vascular system, and
which, according to its entire behavior, is certainly closely related to
hemoglobin. The relation of this pigment to hemoglobin has never been
satisfactorily explained.

Hemoglobin is decomposed by gentle attack into its two components,
globin and hemochromogen. The separation can be effected, for ex-
ample, by adding a few drops of dilute acid to a hemoglobin solution which
is free from salts. Acid hemoglobin is formed as an intermediate product.
It shows an absorption spectrum similar to that of methemoglobin. By
more energetic action of the acid, hemochromogen is split off, but only
when the solution is kept out of contact with the air. In the presence
of air, hematin is always formed; from the latter, by reduction, hemo-
chromogen may be obtained. The digestive juices of the stomach and
pancreas are also capable of effecting this separation of hemoglobin into
its two constituents.

Hematin, whose reduction product, hemochromogen, plays such an
important part in allowing the blood to combine with oxygen, has been
carefully stuched in recent years. Although its constitution has not been
established positively, still we are now able to explain certain relations
existing between it and other compounds of similar construction. The
most important work in this field has been that of Nencki ' and that of
William Kiister.^ According to Kiister, the empirical formula of hema-
tin is C34H34N4Fe05. Nencki and Sieber assume its formula to be
C32H32N4Fe04. The starting-point of these investigations was not
usuall}^ hematin itself, but its hydrochloric acid ester, hemin, or Teich-
mann's crystals as it is sometimes called. Several different formulae
have been proposed for this ester.

It is questionable whether hemin is actually to be regarded as the
hydrochloric acid ester of hematin. Nencki in his work stated that
it was not formed by merely annexing the hydrochloric acid to the
hemaitin molecule, but that there was a replacement of an OH group by
chlorine:

C32H32N4Fe04 + HCl = C32H3iClN4Fe03 + HoO.
Hematin Hemin

' Nencki and Sieber: Arch, exper. Path. Pharm. 24, 430 (1888), and Monatsh. 9,
115 (1888). Nencki and Rotschy: ibid. 10, 568 (1889). Nencki and Zaleski: Z.
physiol. Chem. 30, 384 (1900); Ber. 34, 997 (1901).

= Ber. 27, 572 (1894); 29, 821 (1896); 30, 105 (1897); Z. physiol. Chem. 28, 1
(1899); 29, 185 (1900); Ber. 32, 678 (1899); 33, .3021 (1900); 35, 1268, 2948
(1902) ; Ann. 315, 174 (1900). Z. physiol. Chem. 44, 391 (1905) ; Ann. 345, 1
(1906).

564 LECTURE XXIV.

The iron in hematin may be removed easily by the action of acid. A
product free from iron is thus obtained which is known as hematopor-
phi/rin and is given the formula C16H1SN2O3. Thus, for example, we
may allow hydrobromic acid to act upon hematin:

C32H32N4Fe04 + 2 H2O + 2 HBr = 2 CieHigNaOa + FeBra + Hz.
Hematin Hematoporphyrin

By reduction of hematoporphyrin, mesoporphyrin C16H18N2O2 is ob-
tained, or if the reducing agent is more energetic, hemopyrrole, CgHigN, is
formed. This last substance is methyl-propyl-pyrrole:

HC C — CH2 . C2H5

II II
HC^/C-CHs

N
H

By oxidizing hematin, Kiister obtained two crystalline acids, which he
designated as hematinic acids. One of these is a dibasic acid with the
empirical formula C8H9NO4, while the other is to be regarded as the
anhydride of a tribasic acid. CgHgOs. The formation of these two hema-
tinic acids is illustrated by the following schema:

, CH = C . CH3 ^ CO— C . CH3

hn( I > HN( II

^CH=C . CH2 . CH2 . CH3 ^CO— C . CH2 . COOH

Hemopyrrole or Methylpropylpyrrole Dibasic hematinic acid

CO— C.CH3
0( II

^ CO— C . CH, . CH2 . COOH
Partial anhydride of tribasic hematinic acid.

The last compound, by losing carbon dio.Kide, goes over into the anhy-
dride of methyl-ethyl-maleic acid, C7HSO3.

If we assume that hematin is a simple substance and not a mixture,
and that its transformation into hematoporphyrin takes place quanti-
tatively according to the equation,

C32H32N4Fe04 -I- 2 H2O - Fe = 2 C16H18N2O3,

then it is very easy to go a step farther and assume that hematin is con-
structed of two symmetrical halves connected together by means of an
atom of iron. Now, as we have seen above, hematin and hematopor-
phyrin, by undergoing an oxidation and hydrolysis, both give rise to the
same acids and to the same extent. As these cleavage-products each
contain eight atoms of carbon, it may be safely assumed that hemato-
porphyrin likewise is composed of two equal parts. As was mentioned,

BLOOD AND LYMPH. 565

the hematinic acids are to be regarded as the oxidation products of
hemopyiTole. On this basis we can assign to hemin, the hydrochloric
ester of hematin, the following structural formula:

CH3 . C— C— CH=C(OH)— C = C— CH=CH— C— C— CH3

II II II II II

HC CH 0 FeCl HC CH

\ / II \ /

NH II NH

CH3 . C— C— CH=C(0H)— C=C— CH = CH— C— C— CH3

II II II II

HC CH HC CH

NH NH

The correctness of the above formula has not been established in all its
.details.' It should serve merely to give us an approximate picture of the
structure of hematin. We will state in this connection that the question
has been discussed often, whether the hematin of different species of ani-
mals, or even of animals in the same species, has a uniform composition,
or whether we shall have to assume the existence of different hematins.
This question arose from the fact that different observers claimed to isolate
hemins of different compositions so that the hematins from which they
were made must have been different. The most recent investigations,
however, make it seem more probable that there is but one hematin.^
The observed differences in the composition of hemin may be explained
partly by the different ways in which the substance was prepared, and
partly by the tendency that hemin has of crystallizing out together with
a portion of the solvent.

From the experiments performed in the attempt to explain the consti-
tution of hematin, interesting relations have been discovered between it
and a color-principle, which for a long time has been assumed to exert
quite similar biological functions. We refer to chlorophyll, the pigment
of green plants; although it would be perhaps better to include under the
name chromophyll all the different pigments of the vegetable kingdom
which exert parallel functions. At present, however, chlorophyll, the
green pigment, is the only one of such substances which has been studied
exhaustively. It is hardly to be doubted that the other pigments in the
vegetable kingdom which play the same part in plant economy as that
of chlorophyll have quite similar compositions. We have already
stated that we are not justified in regarding the function of chlorophyll
as parallel to that of hemoglobin, or to hemochromogen. It appears.

■ Cf Lecture XVII, p. 396.

' William Kiister: Z. physiol. Chem. 29, 185 (1900); 40, 391 (1904). K. A. H.
Morner: ibid. 41, 542 (1904).

566 LECTURE XXIV.

on the contrary, that chlorophyll participates in the metabolism of
plants, and especially in the assimilation processes, in a way that finds
no analogy in the case of the pigment of the blood. We may say,
however, that a comparison of the disintegration products of chloro-
phyll with those of hematin, or, better, with those of hematoporphyrin,
shows considerable similarity between these apparently unlike substances.
And yet we cannot rightly draw any conclusions from this agreement as
to the biological uniformity of the pigment of the blood and that of green
leaves. Chlorophyll contains no iron, while in the case of hematin its
functional individuality apparently depends upon the presence of this
element. It is far more fitting to conclude that the close relationship
between chlorophyll and hematin, or its iron-free decomposition product
hematoporphyrin, is explained by the fact that hematin is formed from
chlorophyll. Unfortunately, no one has been able to prove positively
that chlorophyll is actually the mother-substance of hematin. Chlorophyll
is unquestionably transformed to a considerable extent while in the ali-
mentary canal;* apparently various decomposition products are formed.
It is not impossible that the animal organism may make use of these
products for the synthesis of hematin. We make this suggestion because
again and again we are forced to admit that the animal organism is greatly
dependent upon the synthetical work of the vegetable kingdom. To be
sure, the animal cells are capable of effecting comphcated synthesis, but
they require for this purpose building material which has already been
well worked over. The plant cells are capable of producing such material
from the elements. Now hematin is a compound of highly complicated
structure. It is then hardly probable that the animal organism, which
in every other case makes use when possible of the building stones fur-
nished by the vegetable kingdom for accomplishing its synthesis, leaves
this material available for the construction of hemochromogen actually
untouched, and instead effects the complicated synthesis of hematin from
the very simplest material. Here unquestionably is a great gap in our
present knowledge, — a gap which has resulted from the attempt to decide
the question whether inorganic iron compounds or only organic ones can
be utilized by the animal cells for the synthesis of hematin. We have
already shown what a subordinate position is taken by the iron assimila-
tion compared to the formation of the complicated hematin molecule.
We cannot yet decide this question, but desire to leave the impression
that it is by no means improbable that the herbivora obtain in the chlo-
rophyll of their fodder the building material for that component of hematin
which contains the iron; and that the carnivora also utilize the color-
principle of the blood which is contained in their food for the formation of
their own hemochromogen, perhaps, to be sure, only after the hematin in

' Cf. L. Marchlewski: Z. physiol. Chem. 41, 33 (1904).

BLOOD AND LYMPH. 567

the food has undergone a more or less complete preliminary disintegration.
The fact that large amounts of decomposition products of chlorophyll are
found in the fiBces of herbivora, does not in any way speak against the
above assumption. We unfortunately know nothing regarding the extent to
which hematin is destroyed and newly formed in the animal organism. In
the yolk of eggs, and also in the vegetable kingdom, we meet with nuclein-
like substances which, as we have said, are very similar in their elementary
composition to hemoglobin. It is indeed possible that these substances
are used as the raw materials for the hematin synthesis. We do not in
any case need to assume that the animal cell accomplishes the complicated
construction of hemochromogen from simple building material. We only
wish to sound another warning against drawing any conclusions from the
elementary composition of such complicated products. The formation of
hemoglobin, and especially of hematin, in the animal organism is a process
wliich remains absolutely unexplained. The question ^whether iron in an
inorganic or organic condition is assimilated in order to take part in the
synthesis, is far less interesting than that concerning the building material
of the hematoporphyrin. In order to make our position perfectly clear,
we will repeat again that we hold it to be perfectly possible that iron itself,
or in the form of salts, may be utilized as such for combining the two
hematoporphyrin molecules, i.e., in other words the iron is not necessarily
an organic constituent of hematin or of hematoporphyrin, and perhaps,
it can take part in the synthesis only after it has been set free from any
organic compounds which may contain it. A glance at the above tentative
formula of hematin gives us some idea of the separate phases in its
construction.

In the breaking down of chlorophyll, Schunck and Marchlewski ' ran
across a derivative which they called phylloporphijrin. It has the fol-
lowing empirical formula, C16H18N2O. Hematoporphyrin corresponds to
the formula, CieHigNoOs. According to this the two compounds differ
from one another in the amount of oxygen which they contain. March-
lewski ^ showed that both of these substances are to be regarded as dif-
ferent oxidation products of one and the same mother-substance, by
obtaining hemopyrrole, as well as the hematinic acids, from phyllopor-
phyrin. Thereby the close relationship between phylloporphyrin and
hematoporphyrin was established.

Nencld and Zaleski ^ attempted to transform hematoporphyrin directly
into phylloporphyrin. They were able, however, to remove but one of
the hydroxyl groups from the hematoporphyrin. They obtained the
so-called mesoporphyrin which occupies an intermediate position between

' Ann. 278, 329 (1894); 284, 81 (1895); 288, 209 (189.5); 290, 306 (1896).
» J. pr. Chem. 65, 101 (1902).
' Ber. 34, 997 (1901).

568 LECTURE XXIV.

hemato- and phyllo-porphyrin. The following formulae show the close
relationship between these last two compounds:

CH2 CH2

HC— € C(OH)— (OH)C C— CH
II !l I I II II

HC C CH HC C CH

\ / \ / \^ ^ \ / \ /

NH CH2 \/^ CH2 NH

O
Hematoporphyrin: C16H18N2O3.

CH2 CH2
/ \ / \
HC— C CH HC C CH

II II I I II II

HC— C CH HC C CH

NH CH2 \/ CHo NH

0
Phylloporphyrin : CieHigNoO.

Now that we have shown the relations of hematin to chlorophyll, we
shall return to the pigment of the blood and describe the products formed
from its disintegration. While we are not yet in a position to give a
very satisfactory picture of the formation of hemoglobin, or hematin,
we are better informed concerning its decomposition products. The red
corpuscles are without doubt being constantly destroyed, and thereby
hemoglobin is set free. This breaks down, according to our present
knowledge, first into hematin and globin. The latter is probably further
decomposed exactly as is usually the case with proteins. A substance
which for a long time has been recognized as one of the decomposition
products of hematin, is a pigment occurring in the bile which is known
as bilirubin, and has the empirical formula CisHigNaOa which corresponds
to that of hematoporphyrin. That, as a matter of fact, these two com-
pounds ai'e very closely related to one another has been shown by
Kiister,* who obtained from bilirubin, by the same methods that were
used with hematin, two hematinic acids which correspond to those
obtained from hematin. Bilirubin and hematoporphyrin appear to be
isomers. The transformation of hematin into the biliary pigment takes
place in the cells of the liver. If the blood is injected under the skin of
an animal, there will be noticed an elimination of iron at that very spot.
The greater part of the blood's pigment, however, will reach the circula-
tion and be carried to the liver. Here again the iron is first of all

' Z. physiol. Chem. 26, 314 (1898); Ber. 32, 677 (1899); ibid. 35, 1268 (1902);
Z. physiol. Chem. 47, 294 (1906).

BLOOD AND LYMPH. 569

removed. The large deposits of iron in the liver indicate the extent to
which this process has taken place. Bihrubin is not the only pigment
present in the bile. Oxidation products are likewise present which
represent different stages to which bilirubin has been oxidized. We need
not mention their names here, partly because their relations to bilirubin
are not very well known, and partly because it is hard to decide which of
these colored substances are previously formed in the cell and which
result from secondary processes. The best known of these substances is
biliverdin, which may be readily prepared from bilirubin by allowing an
alkaline solution of it to stand in contact with the air. Oxygen is
absorbed, and the solution turns green. One of the tests for the biliary
pigments, the so-called Gmelin's test for bile-pigments, is based upon the
readiness with which bilirubin is oxidized. If a solution of bilirubin-
alkali is cautiously covered in a test tube with a little nitric acid con-
taining some nitrous acid, color rings appear at the contact surface of the
two liquids and in the following order from top to bottom: — green,
blue, violet, red, and reddish-yellow. These different colors are due to
different stages resulting from the oxidation of bilirubin. Before con-
sidering the subsequent destiny of the bile-pigments, we must answer the
question whether the liver is, under normal conditions, the only organ in
which the bile-pigments are formed, or whether it is not perhaps merely
the place where these pigments are eliminated.

Even Virchow ' recognized the fact that peculiar transformations take
place in blood extravasations. The protein component of the hemo-
globin and the remaining constituents of the blood disappear as such,
and there remain beautifully formed crystals of brick-red to ruby-red
color. They are known as hematoidin. This substance contains no
iron, and is considered by many to be identical with bilirubin. According
to our present knowledge, nothing further can be said concerning this
pigment than that it is closely related to hematin and to hematopor-
phyrin. Besides this pigment we will state that pigments containing
iron have also been observed as being formed in the tissues, and that
there has been a constant endeavor to trace a relationship between all
the animal pigments, whether resulting from normal or from pathological
processes, to the blood-pigment. The most pronounced characteristic
that has been noted, however, in every case is as regards the presence or
absence of iron. We would refer merely to what was said in considering
the formation of hematoidin to show that the iron content in no way
indicates the origin of the pigment. Our knowledge in this direction is
still far too limited for us to predict much concerning the relations of the
animal pigments to the other compounds found in the tissues.

Virchow's Arch. 1, 379 and 407 (1847).

570 LECTURE XXIV.

The fact that under certain conditions a compound may be formed from
the blood-pigment in the tissues which is very similar to the bile-pigment
is proved by the discovery of hematoidin. Whether under normal con-
ditions such products are formed in organs other than the liver remains
undecided. In pigeons it has been found that after ligating the bile-duct,
the bile-pigments are found at the end of five hours in the blood-serum.
If at the same time the blood-vessels of the liver are also bound, the bile-
pigments cannot be detected in the blood nor in the tissues after several
hours.' Minkowski and Naunyn ^ arrived at the same result. They
extirpated the liver from a goose, and exposed this goose together with a
normal one to the action of arseniureted hydrogen. Whereas the latter
showed within a short time a copious elimination of urine containing
biliverdin, with the former only hemoglobin was found in the urine.
Such experiments have not yet been carried out with mammals on account
of the great difficulty in completely extirpating the liver from them. We
may safely assume, that with them also the liver is the sole place where
the bile-pigment is formed.

For quite a time this assumption was doubted. It had been observed
that if for any reason the flow of bile to the intestines was prevented,
bile-pigments would appear in the tissues. There is a yellowish coloration
of the skin and of the mucous membrane. The complex of symptoms
which are produced when there is such a stoppage in the flow of the bile,
is known as icterus, or jaundice. Formerly there was a distinction made
between icterus of the above-mentioned etiology, also designated as
hepatogenic icterus, and hematogenic icterus. The reason for this was
because it had been observed that when for any reason there was an
increased destruction of blood-pigment, whether due to the action of
poisons (arseniureted hydrogen, ether, chloroform, toluylene-diamine) or
by infectious diseases, then bile-pigment passed into the urine even
when the flow of bile into the intestines was unrestricted. It was easy to
imagine from this that in such cases the blood-pigment was directly
transformed while in the blood-vessels into bile-pigment. It is not
necessary, however, to make any such assumption. The fact has been
established that intravenous injection of bilirubin causes a considerable
increase in the elimination of bile-pigment in the bile.^ This observation
indicates that the liver also can cause the elimination of bile-pigment,
even when it is circulating in the blood-vessels. Now it is possible
that when there is a greatly increased disintegration of the blood
it may eventually contain so much hemoglobin, and finally so much

' Hans Stern: Arch, exper. Path. Phann. 19, 39 and 42 (1885).
' Ibid. 21, 1 (7) (1886).

5 J. F. Tarchanoff: Pfliiger's Arch. 9, 53 (1874). A. Vossius: Arch, exper. Path.
Pharm. 11, 427 (1879).

BLOOD AND LYMPH. 571

bile-pigment, that it becomes impossible for the liver to talie away
all of it.

Stadelmann ' has shown it to be very probable that in spite of the appar-
ent unhindered passage of bile into the intestine, nevertheless there may
be a stoppage in the flow of the bile. The bile flows under a very slight
pressure, so that the slightest obstruction will stop it, and thereby cause
an absorption of bile by the lymphatics. This may result from a greatly
increased secretion, or from the greater viscosity of the concentrated bile.
At all events, according to these observations we are not justified in
assuming that the transformation of hematin into bile-pigment takes
place in any other organ than the liver.

We shall mention in addition that the restricted flow of the bile
towards the intestines may lead to severe nervous disturbances. Cerebral
effects result, causing delirium, convulsions, coma, and finally death. It
has never been found possible to establish satisfactorily the cause of these
phenomena. It is important to know that they almost invariably result
from chronic obstructions to the flow of the bile. It has been assumed
that the absorbed constituents, and their segregation in the tissues and
blood, were the cause of these severe disturbances. There is no proof of
this, however. We must not forget that where there is a chronic obstruc-
tion to the flow of the bile, it is certain that the metabolism of the whole
liver must suffer as a result. The central position of the liver in the
general metabolism has already been indicated. It is clear that if anyone
of its important functions is entirely abolished, this is likely to affect
the entire organism. On the other hand, the objection may be raised
that the liver can undergo all sorts of severe treatment without necessarily
causing any disturbance in this direction. It may be said, however, that
we are not able to draw conclusions solely on the basis of anatomical
changes concerning the functional condition of a ti.ssue. It is an open
question as to which part is first and most seriously affected. A serious
suppression of metabolism in the cells may take place without there being
any indication of it in the external appearance of the cells or of the con-
tents, and, on the other hand, there may be very great anatomical changes
in a tissue without the functions being other than normal, as long as
the cell complex or the constituents of the cells do not take part in a
pathological process.

In the transformation of hematin into bilirubin, iron is split off. What
becomes of it we do not know. Only a part is eliminated with the bile
itself. It is possible that it is immediately utilized again, or that it
■chooses to be eliminated through the intestines.

The constituents of the bile, especially cholesterol and the bile-

' Arch, exper. Path. Pharm. 14, 231 and 422 (1881); 15, 337 (1882); 16, 118 and
221 (1883).

572 LECTURE XXIV.

pigments, often serve for the formation of concretions and calculi in
human beings. It is not easy to say what causes the formation of
these concretions, which often lead to such severe symptoms. Appar-
ently the primary cause is usually a catarrh of the bile-ducts. Crys-
tals of the above-mentioned substances are then, as a secondary effect,
deposited upon the diseased mucous membrane. Naturally changes in
the conditions of solubility and in the relations of concentration also
play a part in their formation. The pig merit- stones usually contain the
calcium compound of bilirubin. Often mixed stones are found, although
quite frequently we meet with stones of pure cholesterol which show, in a
fractured surface, beautiful clusters of crystals.

The bile-pigments appear in the fseces to some extent as such. For
the most part, they succumb to the putrefactive processes in the ali-
mentary canal, especially those of the large intestine. There is a reduc-
tion of the bilirubin, and urobilin is formed. This last substance may be
obtained directly by the reduction of hematin, or of hematoporphyrin.'
It is also formed by oxidation, when hemopyrrole is allowed to stand in
contact with the air.- Finally, it has been established that by feeding
hemopyrrole to rabbits an elimination of urobilin results. Urobilin,
like hematin, is supposed to contain four molecules of hemopyrrole and
to have the following empirical formula: C32H40O7N4. It is not abso-
lutely known whether urobilin is identical with the so-called hydrobili-
rubin which is obtained by the reduction of bilirubin. Urobilin is
absorbed in the intestine and passes into the urine. It helps to give
to urine its yellow color. Different urobilins have been described as
present in urine, and on the other hand it has even been asserted that
urobilin is not originally present as such in the urine, but is formed by
the action of light. We need not stop to discuss this question, because
at present it is all a matter of conjecture, and we are forced to base our
assumptions in part upon some very doubtful observations. It is ex-
actly the same with the question as to the origin of urobilin. No one
doubts that it is formed in the intestines as a result of putrefaction, but
it is questioned by many that all the urobilin in the urine arises from this
source. Many assume that urobilin is formed outside of the intestines
and in the tissues from bilirubin.

Closely related to urobilin is urochrome, the principal yellow pig-
ment contained in urine. Its chemical nature is but little known.
Uroerythrin also frequently occurs in urine, and is likewise of un-
known composition. It is the red pigment which causes the color
of the sediment in urine, and is also known as Sedimentum later-
it ium.

' Nencki and Sieber: Arch, exper. Path. Pharm. 18, 401 (1884).
2 Nencki and Zaleski: Ber. 34, 997 (1901).

BLOOD AND LYMPH. 573

Small amounts of hematoporphyrin are also present in urine. It is not
without interest that the amount of this substance increases after certain
kinds of poisoning, e.g. lead, trional, sulfonal, and in certain diseases of
the liver. The urine then has the red color of Burgundy.

We have now mentioned what is known of the destiny of the
blood-pigment in the animal organism, and have become acquainted
at the same time with a new, important function of the liver-
cells. We must admit that there are still many gaps which must be
bridged over with regard to our knowledge of the disintegration of
hematin, and there are a great many contradictory results obtained in
the study of the final end-product, urobilin, which remain to be
explained.

It might be expected that some insight into the construction of hemo-
globin would be obtained if we were able to find out where it is formed.
Up to the present time this has not been definitely settled. We are
inclined to believe that hemoglobin is formed at the place where the red
blood-corpuscles come into existence, but even the origin of these cor-
puscles is in doubt. Bone-marrow is chiefly considered in this connection;
and in cases of an unusual new formation of the blood-corpuscles, the ex-
traordinary activity of the cancellous tissue is apparent, even to the naked
eye, by the marked red coloration. It has been asserted frequently that
the spleen is also able to produce red blood-corpuscles, but this asser-
tion has been contradicted. It has been attempted to decide this
question by extirpating the spleen. The operation is withstood quite
well by the organism. While some authors have claimed that there
resulted a marked diminution in the number of red corpuscles, others
were not able to find this the case. Finally, the spleen has been assigned
a part in the destruction of the red corpuscles. Unquestionably this
last assumption is not well founded. To be sure, the spleen usually
contains iron. Experience also teaches us that the spleen is able to
capture foreign substances from metabolism and to retain them, and on
the other hand, by administering iron salts the iron content of the spleen
is considerably inci-eased within a short time, without there being any
evidence of a destruction of red blood-corpuscles. At all events, the part
played by the spleen, either in the formation or the destruction of red
blood-corpuscles, is absolutely unexplained. The same is true regarding
the function of the so-called hernal-lijmph glands which are joined to the
aorta and differ from the ordinary lymph-glands by the fact that the
lymph-passages are either missing or incompletely developed. The
capillaries from the arteries pass directly into the lymph sinus and
into the blood-spaces representing the interfollicular lymphatics, and
from thence the veins lead out directly. The hemal-lymph glands occupy
anatomically an intermediate position between the ordinary lymph-

574 LECTURE XXIV.

glands and the spleen. Their function has never been satisfactorily
explained.'

We must now mention an. important kind of cells which are interme-
diate between those of the blood and of the tissues. The blood does not
communicate directly with the tissue-cells. It neither imparts nutritive
material directly to them nor receives the products of metabolism
directly from them. In this connection the kidneys occupy an excep-
tional position, for in them the blood-vessels of the Glomeruli Malpighi
lie directly at the end of a uriniferous tube. The reason for this is clear:
in this way the waste-products contained in the blood are given up to
the urine as quickly as possible.

In the other tissues of the body, the exchange of material between the
blood and tissue takes place through the lymph. First of all we must
consider the formation of lymph. We may say at once that it has two
sources of material, — the blood on the one hand, and the tissues on the
other. Qualitatively it contains the same substances as the blood-plasma.
Quantitatively, however, there is a difference between the two liquids.
The formation of the lymph, which penetrates into all of the tissues,
has been repeatedly referred to a purely physical process, as, for example,
that of a filtration. We must admit that unquestionably, purely physical
processes do come into play in the giving up of material by the blood
and by the tissues to the lymph, and, on the other hand, in the passage
of material from the lymph either into the blood or tissues.^ We have a
great many reasons, however, for not being willing to admit that our
present knowledge concerning the formation of lymph and all its func-
tions is in accordance with the assumption that only physical forces are
concerned here.

In considering processes which are much easier to understand than
the formation of lymph, we have constantly encountered phenomena
which indicate the existence of a specific activity for every individual
cell, whether it be that of secretion or of absorption. We must admit
that it is far more difficult to study accurately each individual process
when the relations are so complicated as they are in the animal organ-
ism. In every case there are a number of different processes which we
at present cannot examine very closely. One process crosses another.

' See J. Seemann: Die blutbildenden Organe, Ergeb. Physiol. (Asher and Spiro) Jg.
3, p. 1 (1904).

= See Bayliss and Starling: J. Physiol. 16, 159 (1894). W. Connstein: Virchow's
Arch. 135, 514 (1894); Pfluger's Arch. 59, 350 (1899); 59, 508 (1894); 60, 291 (1894);
62, 58, (1895); 63,587 (1896); H. J. Hamburger: Z. Biol. 30, 143 (1893); Arch.
Anat. Physiol. 1895, 281; 1897, 132. R. Heidenhain: Pfluger's Arch. 49, 209 (1881);
56, 632 (1894); 62, 320 (1895). A. Ellinger: Ergeb. Physiol. (Asher and Spiro) Jg.
1 Abt. 1, p. 355 (1902).

BLOOD AND LYMPH. 575

and thus creates new conditions, so that one effect follows another
at the most rapid rate imaginable. It would not be right for us to
be satisfied with the bare explanation that the activity of the cell
is of a specific nature. Of course further investigations must have
free play here. It would be equally wrong to disregard the uncer-
tainty which systematic explanatory experiments encounter. Just as
it is incorrect to maintain that a chemical process prevails in the organ-
ism because it is possible to carry out certain processes in a test tube,
so also we are not justified in assuming that we have established the
fact that a purely physical process takes place because, under certain
definite conditions, we can in this way find a possible cause of a certain
phenomenon. It must be perfectly clear with each advance in the
knowledge of the processes taking place in the animal organism just
where the certainty exists and where it ceases. There is no doubt that
the assumption of a specific secretion being produced by the individual
cells has given rise to an uncomfortable feeling of insecurity in our
entire representations of the life processes. An advance in the science is
only possible when the gaps which still exist in our present information
are circumscribed as sharply as possible. For this reason we shall not
attempt to trace the formation of lymph and its metabolic exchanges
between the blood and the tissue-cells in accordance to physico-chemical
laws.

We should like first of all to abolish any idea that the lymph-stream is
analogous to the blood-stream and carries the substances it contains
from cell to cell. The lymph circulates far too slowly for it to be able
to replenish the cells quickly enough with the material which they need
in cases of active metabolism. We must rather assume that the lymph
of a given organ enters into more intimate relations- between the blood-
vessels and the surrounding tissue. G. von Bunge ' has called our atten-
tion to an observation which well characterizes these relations. The milk
of animals whose young grow rapidly is very rich in lime. The milk of
dogs contains from four to five grams of lime per liter. A bitch weighing
20 to 30 kilograms will secrete half a liter of milk in 24 hours which will
contain from two to two and one-half grams of lime. A liter of plasma
contains only about 0.2 gram of lime, or only iV to tV as much as the same
volume of milk. If, however, the epithelial cells of the mammary glands
must obtain their material for the milk requirements from the transu-
dated plasma, then during the 24 hours, at least 10 liters of plasma must
flow through the mammary glands. This is altogether out of the ques-
tion, for only one or two liters of lymph flows through the entire body in
the course of a day, and far less through the mammary glands. It

Lehrbuch der Physiologie des Menschen, Vol. II, p. 289 (1901).

576 LECTURE XXIV.

follows from this that in the walls of the blood-capillaries of the mam-
mary glands, a liquid rich in lime is secreted, and that, therefore, the
endothelial cells of the capillary walls exercise a selection, as is also the
case with every cell of all living organisms.

It is also possible that we are dealing here with a purely physical
process in which the gland-cells of the mammary gland constantly uti-
lize calcium, i.e., combine with it, and thus cause a continuous dif-
fusion of calcium. But here again we shall have to conjecture how
this may take place. Even the end-products of metabolism are not
carried away, at least not wholly, by a stream of lymph. In this case,
likewise, these products can evidently leave the lymph and penetrate
into the neighboring blood-capillaries and thus be eliminated more
rapidly. The fact that lymph from different places, or even from a
single lymphatic fistula at different times, has different compositions
speaks in favor of such an assumption.

Lymph, as we have said, is qualitatively very similar to the plasma.
It contains the same substances. Of especial interest is its content of
fibrinogen and fibrin-ferment. The amount of each of these substances is,
however, very slight. Lymph coagulates slowly, and not all at once.
Serum-albumin and serum-globulin are the two principal proteins which
have been found in it. Lymph also contains cellular elements, especially
the leucocytes, in varying numbers. Unfortunately, we are not very well
informed concerning the composition of the tissue-lymph and that of
the lymphatics. Most of the analyses that have been made are of lymph
obtained from the thoracic duct, which contains chyle. For this reason,
we are able to say but little concerning the formation of lymph and its
dependence upon definite conditions. On the other hand, the work of
Asher ' and his collaborators has served to explain the relations between
the amount of lymph formed and the work of certain organs. The influ-
ence exerted by the work of an organ is best illustrated in the case of the
salivary glands. If a strip of blotting paper moistened with vinegar is
placed in the mouth of a dog, there is a copious secretion of saliva, and at
the same time there is an increased flow of lymph from the lymphatics
of the neck. This increased flow of lymph does not depend upon a corre-
sponding increase in the rate of flow of the blood. In muscular work,
likewise, it may be shown that there is an increased formation of lymph.

We must consider, here, an important discovery, namely, that there
are certain substances which incite the lymph flow. These are known as
lymphagogues, and are of two kinds. The lymphagogues of the first group
are obtained from the extracts of crab-muscles, blood-leeches, anodons,

■ Asher and Barbera: Z. Biol. 36, 154 (1897); 37, 261 (1898); Asher and Gies:
ibid. 40, 180 (1900); Asher and Busch; ibid. 40, 333 (1900); Asher and Barbera:
Zentr. Physiol. 11, 403 (1897).

BLOOD AND LYMPH. 577

from the liver and intestines of dogs, and from strawberry-extracts, etc.
In this case, the increased flow of the lymph can also be attributed to the
increased activity of an organ, the liver especially. On the other hand, it
has been assumed that these substances act upon the endothelium of the
capillaries in such a way that they are incited to increased activity. To
the lymphagogues of the second group belong sugar, urea, common salt,
etc. These cause an abundant lymph formation, whereby the plasma,
as well as the lymph, becomes more dilute. In this case also a specific
activity of the cells is presumed; here, that of the tissue cells.

It is not difficult to understand that the work of organs increases the
production of lymph; for, on the one hand, the cells of the active organ
require an increased supply of nutriment, and, on the other hand, they
yield an increased amount of metabolic end-products.

One might be tempted to ask why the presence of the lymph and the
liquid in the tissues is at all necessary, and why it would not be better to
have a more direct interchange between the blood and the cells of the
tissue. The utility of this arrangement is very clear; for, in the first
place, it is evident that by the interposition of a liquid which penetrates
into the smallest spaces between the tissues, it is made possible to effect a
more delicate exchange of material; and, on the other hand, it prevents
the cells from being over-supplied with nutriment at any one time; and,
furthermore, by this means it is possible to keep the composition of the
blood fairl}^ constant, which would not be the case if the end-products of
the metabolism in an active organ were given up to the blood all at once.
The Ij'mph also serves as a diluting medium. An observation of Asher
supports this view. He showed that normal Ij'mph contains products
which, if directly introduced into the blood circulation, would give rise
to disturbances. If, for example, some of their own lymph from the neck
be injected into the internal carotid artery of dogs, changes are at once
produced in the blood pressure.

The organism also has a means of protection in the so-called lymphatic
glands, or lymph-nodes, which are situated in the course of a lymph-vessel.
They have various functions. According to the way they are constructed,
it is easy to imagine that they, to a certain e.xtent, act as filters and keep
back certain substances which are injurious to the body. It is also con-
ceivable that the}' are able to combine with substances which are given
up to the lymph in large amounts. They are constantly giving up leuco-
cytes, so that they in this way take part in the general metabolism.

Closely related to the lymph are certain liquids which are secreted by
the serous membranes, which are provided with an endothelium and fulfil,
for the most part, purely mechanical functions. These liquids are called
transudates. Under normal conditions there is but a small amount of
these. Thev are deficient in formed elements. .•Is regards the formation

678 LECTURE XXIV.

of the transudates the question has been much discussed whether they
result from a filtration from the blood-vessels or whether they represent an
" active" secretion. Here the relations are somewhat similar to those of the
lymph. The fact that transudates contain the same substances as plasma,
and, with the exception of albumin, in about the same proportion, cannot be
regarded as an absolute proof of a filtration having taken place. Under
pathological conditions the amount of transudate may increase enormously.
If this formation results from an inflammatory process, it is called an
exudate. The exudates are richer in cellular elements. If the amount
of the latter is greatly increased, we called the liquid pus.

Transudates are found normally in the sac of the pericardium, between
the layers of the pleurae, and in the peritoneum. To this class of liquid
belongs the cerebrospinal fluid and perhaps also the aqueous humor. A
very similar liquid is found around the joints and in the bursce mucosee
which is known as synovia. It contains a substance similar to mucin.
The true transudates are composed, as we have said, of the same con-
stituents as plasma, and it is noteworthy that they contain fibrinogen,
but hardly to an extent sufficient to permit spontaneous coagulation.

If we examine more closely the relation between the lymph and the
blood, we shall arrive at the conclusion that, to a certain extent, all the
cell-elements of the animal tissues are either directly or indirectly mois-
tened by these fluids. The tissues no longer appear to us as rigid struc-
tures, as it is customary to consider them from an anatomical point of
view. There are no sharp lines drawn between the blood, lymph, and
the body-cells. There is never any repose. A stream of blood continu-
ally flows towards the cells, and conversely, the cells by the aid of the
lymph send their products to the blood, and thus all the different ele-
ments combine to form a physiological unit for each individual organ, or
perhaps only for definite cell-groups. We can now understand what
great difficulties are met with in the attempt to trace experimentally the
course of a reaction in the animal organism. The lymph forms on the
one hand an intimate means of communication between the blood and
the tissue-cells, and on the other hand it serves as a barrier between
them. Substances administered, whose participation in cell-metabolism
we should like to be able to trace, may reach only as far as the lymph, and
may be excluded from the metabolic processes of the cell itself. Whether
transformations take place in the lymph, or whether the lymph modifies
the products obtained from the blood and prepares them so as to meet
the existing demands, is something of which we have no information.
Here a great unexplored field lies before us.

LECTURE XXV.

THE ELIMINATION OF METABOLIC PRODUCTS FROM THE

BODY.

We have already called attention to the important fact that the blood,
and especially the plasma or the serum, has a remarkably constant
composition. This holds true at least within certain limits, and in as
far as we can detect by means of our faulty methods of examination.
This relative constancy holds not only, as we have shown, with regard
to the quantitative relations in which the substances are present, but
also, as far as we are able to see, for the qualitative relations. Thus we
feel justified in assuming, for example, that the amount of protein in the
blood may indeed vary somewhat, but it does not change its nature in
spite of the most varied forms of nourishment.' We intentionally speak
of a relative constancy, because there can be no such thing as an absolute
constancy in the composition of the blood, for from moment to moment,
according as this or that organ comes into action, the blood is bound to
receive quite different metabolic products. Such products are, however,
present in such a state of dilution that we can detect them only in a large
quantity of blood. Again, during the digestive process and when the
absorption and assimilation is at its height, sometimes this substance and
sometimes that one will circulate in the blood to an increased extent.
As a rule, however, these differences are so slight that they cannot be
detected bj' the present methods of analysis. They are usually concealed
by experimental errors. At the same time it is perfectly evident that
the composition of the blood is regulated to a remarkable extent and
kept constant within narrow limits. There are many ways in which
this regulation is effected, and they are not all of the same nature as is
usually assumed. First of all, the blood is kept from being flooded with
substances foreign to it, as they are contained in the food, by the activity
of the intestine. Were it not for this, we could not understand, as we
have repeatedly stated, why the composition of the serum should always
remain qualitatively and quantitatively practically the same. By means
of the activity of the digestive ferments, all of the complicated and
widely different nutrient substances, which vary from day to day, are

' Abderhalden and Samuely: Z. physiol. Chem. 46, 193 (1905). Emil Abderhalden:
Zentr. Stoffwechsel- und Verdauungskrankheiten 5, 647 (1904).

579

580 LECTURE XXV.

disintegrated; and from the resulting products the intestines, and per-
haps the liver as well, forms substances suitable for the body. The
intestine assumes by this function a characteristic position. It takes
care that the tissue-cells always receive the same nutriment, and makes
them absolutely independent of the nature of the food which is eaten.
We cannot be far wrong in ascribing to the intestine in this sense an
important part in the maintenance of the individuality of each species of
animals.

A second mechanism for regulating the composition of the blood is
found in the lymph. The exchange of material between the blood and
tissue-cells takes place, as we have seen, through the lymph. The lymph
receives substances from the blood which the cells of the tissues require,
and on the other hand it gives up to the blood the waste-products which
it receives from the tissue-cells. It is able to retain these last-mentioned
substances for quite a time, only gradually giving them up to the blood,
for further elimination from the body.

The chief organs for the elimination of the metabolic end-products,
and of the substances which the organism cannot utilize, are the kidneys.
They guarantee the maintenance, as far as possible, of the blood-uni-
formity. Under normal conditions they are perfectly adequate for
this purpose. If it happens that the kidneys, for some reason or other,
are not able to remove all of the foreign substances from the blood,
then other organs attempt to act as their substitute. Most of all the
different glands contained in the organism may be active in this sense,
and even under normal conditions there is no doubt that small amounts
of the waste-products are eliminated in this way. This is best shown
by introducing into the body substances which are foreign to it. Thus,
if we introduce potassium iodide into the intestines, some of it will soon
appear in the saliva and in the sweat. If morphine is injected subcu-
taneously, a part of it is eliminated in the stomach. Urea is likewise
found in sweat, particularly when the kidneys are not adequate to the
demands placed upon them. The intestines also form an important organ
for elimination, and normally. We have already seen that the alkaline
earths and heavy metals are unquestionably largely eliminated directly in
the intestines, and in fact chiefly through the rectum. The animal organism,
furthermore, is able to combine many of th,ese foreign substances together,
whereby the blood and the tissues are prevented from being flooded with
them. Such substances may then be eliminated gradually in the course of
several weeks. We have also repeatedly called attention to the ability
of the tissue-cells, and especially of the liver, to make many substances
harmless by oxidizing or reducing them, and in some cases conjugating
them with certain substances, such as glycocoll, glucuronic acid, sulphuric
acid, or urea.

THE ELIMIXATION OF METABOLIC PRODUCTS. 581

The importance of the other glands, large and small, as organs for the
elimination of substances foreign to the organism and of the end-products of
metabolism is insignificant compared to that of the kidneys. Their anatom-
ical structure characterizes them for the exercise of their most important
function. In the first place we notice the peculiar nature of the blood-
vessels. The arteries form branches and side twigs; each of the afferent
vessels terminates in a globular bunch of capillaries, the glomerulus or Mal-
pighian tuft. The blood leaves the glomerulus through a so-called efferent
vessel, or Fas efferens, which also breaks up into a close capillary plexus
which surrounds the secreting tubes. From this network come venous
radicals, which empty into the veins of the kidneys, and through which
the blood, which has meanwhile been freed from the metabolic end-products
and other waste-material, leaves the kidneys. The termination of the
afferent vessels in the Malpighian tuft has awakened the most interest.
It is worth mentioning that these vessels are considerably narrower than
the corresponding efferents.

The Malpighian tuft is within the so-called Bowman-Muller capsule.
This consists of a thin pouch consisting of epithelial cells, into which, as it
were, the tuft has been pushed. It represents the beginning of a uriniferous
tube. The latter are not simple drainage channels, but follow, first of all,
a tortuous path, the. first convoluted tube, or tubulus contortus. Then the tul^e
narrows suddenly and describes a loop, reaching into the medulla, known
as Henle's loop. The tube then turns back towards the cortex, forming
an irregular convoluted tube, that of Schweigger-Seidel, which passes
through a narrower arch into the straight collecting tube. Several tubes
which have up to this point been entirely independent, empty into this
collecting tube. It terminates, together with other similar tubes, at the
surface of a papilla in the calyx of the kidney. We will state, moreover,
that the epithelium of these different parts of a uriniferous tube is not
uniform. We mention briefly these relations, in order to show that the
process of forming a secretion by the kidneys is by no means a very simple
process. There is some reason for the complicated construction of the
kidneys. In considering the function of the kidneys, we must hold close
to the anatomical relations, and attempt to explain the significance of the
differently organized parts of the uriniferous tubes. Before taking up the
question of the secretion of the urine, we will briefly mention, in connection
with the above brief description of the construction of the uriniferous
tubes, those researches which have been undertaken in the attempt to
decide what the function of each different part of the tube is. Right at
the start it may be stated, that, according to all we now know, the urine
is not eliminated from the blood in the form in which it eventually reaches
the calyx to be emptied into the bladder. There is probably an absorption
of substances, partly of water and partly of other substances, while it is in

582 LECTURE XXV.

the tubes.' To l)e .sure, there are a number of experiments which do not
agree with such an assumption.^ A reabsorption has been observed
only under conditions which cannot be regarded as normal. The frog is
a particularly suitable subject for such experiments. In this animal the
kidneys receive their blood from two sources, the renal artery, and the
renal portal vein. The first provides the Malpighian body with ma-
terial, while the latter leads directly to the uriniferous tubule. If the
renal artery is ligated, the secretion of urine stops completely. On
the other hand, if the flow of blood through the renal portal vein is
stopped, then urine continues to be secreted. If the uriniferous tubules
have the function of taking up water and solid constituents from the
products secreted by Bowman's capsules to give them up to the blood
again, then it would be expected that after ligating the blood-vessels
supplying the uriniferous tubule, that there would be an increased
elimination of urine. This was, however, not the case. On the con-
trary, the amount of urine diminished. We must regard this question
as unsettled. It is indeed conceivable that an absorption takes place
only under certain conditions. We must at this place mention how
extremely difficult it is to obtain a clear judgment when there is any
meddling with the normal functions of the kidneys. We never know
exactly what the primary cause of a phenomenon is, and what takes place
only secondarily. Above all, we have to consider the important influence
of the blood-supply upon the formation of the urine. It is, for example,
perfectly possible that in the above case the diminished secretion of the
urine may have been caused by an obstruction in the flow of the blood to
the glomeruli. We shall come to this question again when we discuss
the question of reabsorption.

In order to investigate the functions of the separate divisions of the
uriniferous tube, substances have been introduced into the circulation
which can be easily detected in microscopical preparations. Thus
Heidenhain ' found that after the injection of sodium sulphindigotate
into the blood, it reappeared in the epithelial cells of the uriniferous
tubules. He concluded from this discovery that these cells have the
function of adding certain specific constituents of the urine to the secre-
tion which it receives from the capsules of Bowman. This is not neces-
sarily true, for the microscopical pictures might equally well have
been caused by a reabsorption of the dye from the uriniferous tubule.
Carmine has also been used for such experiments,* and the results of

' H. Ribbert: Virchow's Arch. 93, 169 (188.3). W. M. Sobieranski: Arch. e.xper.
Path. Pharm. 35, 144 (1895).

' A. Gurwitseh: Pfluger's Arch. 91, 71 (1902). A. P. Beddard: J. Physiol. 28, 20
(1901).

= R. Heidenhain: Arch, mikro. Anat. 10, 1 (1874). Cf. Pfluger's Arch. 9, 1 (1874).

* Cf. Adolf Schmidt: Pfluger's Arch. 48, 34 (1891).

THE ELIMINATION OF METABOLIC PRODUCTS. 583

these experiments also are capable of more than one interpretation.
Dreser ' attempted to find out at which place acid-fuchsin was elimi-
nated. He injected daily three or four cubic centimeters of acid-fuchsin
into the dorsal lymph-sac of frogs. An hour or so afterwards there was
no pigment present in the glomeruli nor in the convoluted tube, but only
in the lumen of the central part of the tube. If the injection were
repeated, the glomeruli remained colorless; but in the convoluted tubes,
the red coloration gradually extended into the end of the epithelium
which is toward the lumen. Experiments with alizarin-carmine showed
that the coloration did not appear in the Tubuli contorti, but only in the
distal parts of the tubes. Dresser concluded from these experiments and
those with other dyes, that in the convoluted tubes secretion alone takes
place, and no reabsorption. We see, therefore, that the decision as to
the absorption capacity of the epithelium of the convoluted tubes is entirely
dependent upon the interpretation of these microscopical pictures. As
long as this problem cannot be decided positively, it is quite impos-
sible to establish the function of the different parts of the uriniferous
tubes. The same microscopical appearance may be regarded as resulting
from an absorption or from an elimination. The entire question as to
the special functions of the anatomically different parts of the uriniferous
tubes remains absolutely unsettled by the above e.xperiments.

Let us now see whether our knowledge of the functions of the kidneys
is sufficient to give us a clear idea of the formation of urine. In the first
place we must state that it has never been found possible to detect posi-
tively the presence of secretory nerves in the epithelium of the kidneys.
All the nervous influences which have shown an effect upon the elimina-
tion of urine can be either directly or indirectly attributed to a change
in the innervation of the blood-vessels. The amount of the blood circu-
lating is closely related to the amount of urine secreted. This relation is
almost self-evident, for the greater the amount of blood passing through the
kidneys, the greater will be the opportunity for the cells of the kidney
to form their secretion. The blood-pressure also is to be considered.
The whole arrangement of the glomeruli is such that the blood must pass
the Malpighian body with a relatively high pressure. In this respect the
above-mentioned behavior of the efferent vessels is important, which are
considerably narrower than the afferents. Ludwig ^ makes use of this
fact for the foundation of his much-discussed theory of urine-elimination.
He attempted to make his explanation as mechanical as possible, and
assumed first of all that there was a filtration through the glomeruli at

' H. Dreser: Z. Biol. 21, 41 (1885). Cf. P. Griitzner: Pfliiger's Arch. 24, 441
(1881). M. Nussbaum: ibid. 16, 139 (1878); 17, 580 (1879).

- Ludwig: Wagner's Handworterbuch der Physiol. 2, 629 (1844); Wiener med.
Wochenschr. 14, No. 13, 14 (1864); Sitzber. Kais. Akad. Wissensch. 48 (1863).

584 LECTURE XXV.

the beginning of the uriniferous tubes. According to this, the urine
would show a very similar composition to that of the plasma of the blood.
This, however, is not the case. Ludwig furthermore assumed that there
was a re-absorption in the uriniferous tubes, both of water and of dis-
solved substances. Very soon, however, important objections were raised
agairLst this theory of a filtration of all of the constituents of urine.
Above all, it was pointed out that while it was comprehensible that the
proteins in the blood could not pass through the vascular endothelium, on
the other hand it would be expected that certain other substances, for
example sugar, which is always present in the plasma, would filter through
into the urine. This is, however, not the case under normal conditions,
and the same is true of certain other substances. To account for this it
has been suggested that perhaps the sugar may not circulate as such in
the blood, but that it is combined with some other compound which
will not filter through the medium. There is no support for any such
assumption. The quantitative composition of the urine also speaks
against any such simple filtration process taking place in the formation
of urine. The plasma contains about 0.05 per cent of urea. We would
then expect the urine to contain about the same amount of this sub-
stance, whereas in fact about 2 per cent is usually present. If we are to
retain the idea of a pure filtration, then we must necessarily assume
that the urine is concentrated in the uriniferous tubes to about jV of its
original volume. It has, furthermore, been found that if the supply of
blood to the kidneys is entirely stopped, that the secretion of urine
ceases; but, on the other hand, when the blood is allowed to flow again
through the kidneys, it is about 45 minutes before the secretion of
urine begins. If we believe that the endothelium of the glomerulus
and that of Bowman's capsule is a mere filtering membrane, it is hard
to account for the long cessation of its function.

Especially quite recently, more and more facts have become known,
which indicate that the kidneys act analogously to other glands during
the formation of their secretion. For one thing, it has been observed
that an increased secretion causes a rise of temperature, and that when
there is more work done by the kidneys, there is more oxygen consumed
and more carbonic acid produced.' Our present knowledge teaches us
that we cannot be far wrong in assuming that practically the whole
function of the kidneys is analogous to that of the other glands and that
no simple filtration ^ can take place; both the epithelium of the glome-
rulus, as well as that of the uriniferous tube, probably produce an actual
secretion. We know of various substances which increase the secretion

' J. Bancroft and T. G. Brodie: J. Physiol. 32, 18 (1904).

2 Cf. Torald Solhnann; Ann. J. Physiol. 13, 241 (1903). W. Filehne and J. Biber-
feld: Pfluger's Arch. Ill, 1 (190G).

THE ELIMINATION OF METABOLIC PRODUCTS. 585

of urine, just as we have met with such substances in the study of other
glands. To be sure, it has been shown that a great many of these sub-
stances are capable of exerting only indirectly an influence upon the
kidney-cells, by affecting the flow of the blood through the kidneys on
the way to the vascular innervation.' For other substances, the result
cannot be traced to this cause.

In order to prevent misunderstandings, we will at once state that purely
physical processes undoubtedly do play an important part here as in the
case of all absorption and secretion processes in the animal organism.
It is perfectly possible that a pure filtration process may act in conjunction
with other processes in the formation of urine. At the same time it would
be wrong to assume that the existence of a filtration proce.ss is proved.
We can only infer that it takes place, and are at once compelled to make
the auxiliary hypothesis of the reabsorption by the epithelium of the
uriniferous tubes. Another possibility seems to us as far more probable,
namely, that the Malpighian tuft is not the sole place where the constituents
of the urine are secreted. We have already seen that the efferent vessels,
after emerging from the tuft, again break up into a capillary network,
which surrounds the secreting tubes in the cortex. This behavior of the
blood-vessels must surely be of some significance in the formation of the
urine. It is possible that a back-absorption takes place here, but it is also
conceivable that the epithelium of the uriniferous tube is only able to
withdraw definite substances from the blood, concentrates them, and
finally sends them on at different periods towards the lumen of the tubes.
First of all, we must remember that the Bowman's capsule possesses numer-
ous finely branching nerve-fibers, which originate in the vasomotor nerves.
The blood-capillaries, also, are abundantly supplied with nervous plexuses.
Furthermore, it has been found that nervous branches exist which supply
the uriniferous tubes; and in fact we see separate plexuses of non-
meduUated fibres, arising especially from the Tubuli contorti. Such
nerve-endings have also been observed for the epithelium of the Bow-
man's capsule, for the straight tube and also for the collecting tubes.
The fact that the vessels of the kidneys, and especially those of the
capillaries of the cortex have such an extensive innervation, suffices to
account for the sensitiveness of the renal vessels to all sorts of different
influences. More and more it has become evident that a great number
of those substances to which has been ascribed a specific action upon
the parenchyma of the kidneys, only influence the formation of urine by
accelerating or retarding the flow of the blood. The intimate dependence
of the secretion of the kidneys on the blood-supply may have been the
chief reason for assigning to the kidneys a position different from that of
the other organs of the body.

' Cf. O. Loewi: Arch, exper. Path. Phann. 53, 15, 3.i, and 49 (1905).

586 LECTURE XXV.

We have repeatedly learned that other glands are to a certain extent
independent of the blood-supply. Thus the pancreas is constantly forming
its secretion, but gives it up only after certain kinds of stimulation. It is
otherwi.se with the kidneys. Their activity is different, because their func-
tion has an entirely different significance from that of all the other glands.
The kidneys must be very delicately adjusted to the composition of the
blood. They are obliged to work verj' rapidly in all cases, and are not
obliged in every case to follow stimulations which are communicated
to them reflexively by the nervous system. The chemical nature of the
blood invariably has an influence. Now, is this because the vasomotor
nerves are directly influenced by the composition of the blood, so that, for
example, an enlargement of the ve.ssel or restriction of it will be effected,
or because certain components of the urine act directly upon the epithelium
of the uriniferous tubes? We hold that the last assumption is very prob-
able, and imagine that certain specific substances are captured by this
epithelium, which are concentrated and then given up again. Only in
some such way as this are we able to account for the relatively high con-
centration of the urea. There are quite a number of different observations,
which indicate such a specific function of the kidney epithelium. A few
examples will be cited. The elimination of uric acid has been studied
most closely, and its presence is easy to detect.' If, for example, a solu-
tion of uric acid in piperazine is injected subcutaneously into rabbits,
there takes place first of all a considerable diuresis. In from twenty
minutes to an hour uric acid may be detected in the tubes of the med-
ulla. The glomeruli and Bowman's capsules are perfectly free from
deposits of uric acid; but, on the other hand, the epithelium of the con-
voluted tubes contains granules of uric acid, ami chiefly in the end of the
tubes facing the lumen. Anten ^ obtained corresponding results in the
kidneys of dogs. He cut out the kidneys from the general circulation of
a live dog, and then passed a solution of freshly-precipitated silver chloride
in ammonia through the organs, in order to precipitate the uric acid present
as silver vu-ate. The kernels of the silver salt were found chiefly in the
cells of the convoluted tubes, and particularly at the basal part of the
cells. The epithelium of the ascending tube of Henle's loop also showed
isolated accumulations, but this was not the case with the descending part
of the loop. One might naturally be inclined to object that the appear-
ance noted might result from a reabsorption just as well as from a secre-
tion of the uric acid. There are, however, so many observations ^ of

' Cf. Sauer: Arch, niikros. Anat. 53, 21S (1899). W. Ebstein and A. Nicolaier: Ex-
perimentelle Erzeugung von Harnsteinen, Wiesbaden, 1891, and Virehow's Arch. 143,
337 (189G). O. Minkowski: .A.rch. exper. Path. Pharm. 41, 375 and 410 (1898).

' Henri Anten: .\rch. internat. de pharmacodynamie et de therapie, 8, 455 (1901).

' Cf. Courmont et Andr^: J. physiol. et pathol. general, 7, 255 (1905).

THE ELIMINATION OF METABOLIC PRODUCTS. 587

this nature made under quite different conditions that we may well
assume that an actual secretion of uric acid takes place, and its separa-
tion from the blood is probably the result of a selective action of the
.epithelium of the above-mentioned portions of the uriniferous tube. We
will mention in addition that Hober and Konigsberg ' have proved that
the epithelium of the uriniferous tube is not only able to take up colors
which are soluble in lipoids, but also those which are insoluble in lipoids.
Unfortunately, it has up to the present not been found possible to local-
ize the secretion of urea and other substances in the same way as in the
case of uric acid.

We do not in any way intend to suggest that the secretion of the urine
is a perfectly simple and uniform process. Unquestionably a great num-
ber of different processes are taking place side by side, which mutually
assist one another. On one side constituents of the urine are given up
through the ]\Ialpighian bodies to the capsules of Bowman, and 'evi-
dently at this place the greater part of the water is sent out, while the
epithelium of the uriniferous tube is constantlj' removing definite con-
stituents from the blood, accumulating them and then giving them up
again inward to the lumen of the tube. If we consider in addition that
many observations indicate that there is more or less al^sorption in the
uriniferous tube of some of the substances which have previously been
secreted, of water especially, then we shall begin to understand that
diuretics can find a number of different points of attack, and cause, in a
number of different ways, disturbances in the secretion of the urine.

We must come back once more to the fact that under normal condi-
tions there is no sugar in the urine. As we have said, it has been sug-
gested that this was because the glucose did not circulate as such in the
blood, but combined in some way with colloidal substances, although
direct experiments' have shown that this representation is in no way
justifiable. Sugar is present as such in the blood. Evidently the vas-
cular endothelia of the kidneys are adjusted to a certain definite sugar
content of the blood. When more than this is present the sugar passes
over into the urine. Now it seems probable that certain substances cause
sugar to pass into the urine even when there is no glucohemia. Such,
for example, is phloridzin, which, according to many observers, acts
directly upon the kidneys, or indeed at first upon the vascular endo-
thelia.^ Recently Underbill and Closson ^ have stated that those forms
of glucosuria, which appear when common salt is introduced into the
circulation, are in some cases to be regarded as due to a direct influence

' Hober and Konigsberg: Pfluger's Arch. 108, 32.3 (1905).

' Leon .\sher and R. Rosenfeld: Zentr. Physiol. 19, 449 (1905). See Lecture H, p. 30.

' See Lecture V, p. 81.

* Am. J. Physiol. 15, 321 (1906).

5SS LECTURE XXV.

upon the kidneys. They found that a great deal depended upon the
place in which the salt solution was introduced. If it was an artery of
the brain, glucohemia ensued, and consequently glucosuria, but no poly-
uria.' When, however, they injected the salt solution into one of the
veins of the body, polyuria soon resulted, and at the same time an elimi-
nation of sugar took place, but, instead of the amount of sugar present
in the blood increasing, it decreased. This case of glucosuria, therefore,
must arise from another cause, and may be attributed to an action upon
the endothelia of the kidneys.

If we consider that the kidneys have the function of removing all
abnormal substances from the blood, and any exce-ss of normal ones, then it
is self-evident that definite statements cannot be made concerning the
composition of the urine. It is dependent first of all upon the nature of
the nourishment and upon the intensity of the metabolism of the cells.
There is nothing uniform concerning the amount of urine eliminated in a
day, nor concerning its reaction or other behavior. The individual
products which are eliminated in urine we have already discussed, and,
in each separate case, traced the product to its source. The end-products
of metabolism are always eliminated with a greater or less quantity of
salts. These originate, to be sure, partly from decomposition and partly
from destruction of tissue, but for the most part they may be traced to
the food itself. The amount of water in urine does not depend entirely
upon the amount that is drunk, but is materially affected by the amount
that is utiHzed in the organism. We shall soon see that by the evapora-
tion of water from the surface of the body, the animal organism has a
very efficient means of regulating its temperature. We may expect
that one and the same individual, under conditions remaining constant
and a diet which is qualitatively and quantitatively the same each day,
would eliminate a urine which would show a constant composition within
narrow limits. It is remarkable that but few exact and complete analy-
ses of urine have ever been made. Usually the composition of the
food that is eaten is entirely disregarded. It is clear that such analyses
are useless for drawing any conclusions or for future inquiry. The great
gap in our knowledge is thus made more apparent, for we would unques-
tionably be able to draw certain conclusions as to the metabolism of the
cells if there were exact analyses which took into consideration all the
substances present in urine, and such investigations would be of great
help in the case of pathological processes. Quite recently Otto Folin ^
has imdertaken the analysis of urine from a single individual during
several days in which the diet remained the same. We regret that we
cannot give all the values he obtained in his work, but we have to be

' See Lecture V, p. SI.

= Am. J. Physiol. 13, No. 1, 45 aud 06 (1905).

THE ELIMINATION OF METABOLIC PRODUCTS.

5S9

satisfied here with certain parts of it which are shown in the following
summary. The nourishment consisted of 500 c.c. milk, 300 c.c. cream
with a fat-content of 18 to 22 per cent, 450 grams egg, 200 grams Hor-
Hck's Malted Milk, 20 grams sugar, 6 grams salt. This mixture con-
tained about two Uters of water, and in addition 900 c.c. were drunk.
In this food there was contained 119 grams albumin, about 148 grams
fat, and 225 grams carbohydrate. The mixture was shown to have a
constant composition by the determination of the chlorine from day- to
day (about 6.1 grams), of the sulphuric acid (about 3.7 grams), of the
phosphoric acid (about 5.8 grams), and of the nitrogen (about 19.0 grams).

ANALYSIS OF

THE URINE OF

A NORMAL INDIVIDUAL

Body

Volume

Total Ni-

Nitrogen

Nitrogen

Nitrogen

N itrogen
as Uric

Nitrogen
in Other

Weight.

trogen.

as Urea.

monia.

Acid.

pounds.

kg.

cc.

70.8

Sept. 21

1520

15.9

13.7

0.64

0.61

0.08

0.81

Sept. 22

1530

16.6

14.5

0.72

0.58

0.10

0.80

Sept. 23

1460

16.6

14.4

0.73

0.56

0.11

0.83

Sept. 24

1430

16.5

14.2

0.75

0.52

0.12

0.90

70.1

Sept. 25

1380

16.6

14.5

0.86

0.54

0.11

0.85

Each 100 grams of the total nitrogen

were distributed

as follows:

Date.

Urea.

Ammonia.

Urea and
Ammonia.

Creatinine.

Uric Acid.

Nitrogen
in Other

Com-
pounds.

September 21

September 22

September 23

September 24

September 25

85.9
86.9
86.5
86.1
85.7

4.1
4.3
4.4
4.5
5.2

90.0
91.2
90.9
90.6
90.9

3.8
3.6
3.4
3.2
3.3

0.5
0.6
0.7
0.7
0.7

5.7
4.6
5.0
5.5
5.1

Neutral

Total Sul-

Inorganic

Ether-sul-

Sulphur

phur De-

Sulphuric

phuric

Deter-

Date.

termined
as Sul-

Acid.

Acid.

mined
as Sul-

phate.

(SO

(S.)

phate.

(S3)

3.31

3'35
3.20
3.25

2.85
3.00
2.89
2.73
2.92

0.25
0.25
0.28
0.24
0.21

0.21

September 23

September 24

September 25

0.18
0.13
0.12

590

LECTURE XXV.

Date.

In Per cent of the Total
Sulphur.

Acidity in

c.c. of

10

Solution.

Si

Sj

S3

Total.

Inorganic.

Organic.

September 21

September 22

September 23

September 24

September 25

86.1

86.3
85.3
89.8

7.6

8^3
7.5
6.5

4 1
3.7

589
630
625
617
646

219
299
432

276

370
331
193

370

September 21
September 22
September 23
September 24
September 25

Total
Phosphate
as PjOs

3.98
4.16
3.84
3 68
3.85

Chlorine

in Grams.

6.3

5.7
5.8
5.7
5.2

Indican

(Fehling's

Solution

= 100).

140
150
140
140
130

It is evident from these values that the urine during these five days
showed a very constant composition. It would be very interesting to
carry out such experiments with a uniform diet, and especially with the
S£ime kind and amount of albumin, also on a large scale during patho-
logical conditions. In such a way we should obtain an insight into the
cell-metabolism under different conditions. At the same time it is not
true that we can draw very far-reaching conclusions in all cases as to the
cell-metabolism from the composition of the urine. We must always
remember how little we know concerning cell-metabolism and of the
dependence of one organ upon another. It is indeed possible that
there is an exchange of material in such a way that the decomposi-
tion-products from one organ are utilized by another. Thus there may
be a considerable destruction of tissue of certain specific composition,
which would not show any indication in such an analysis, because there
might not be any of the products from the destruction of this tissue,
in the urine. The kidney may be an economizing organ of the animal.
It is perfectly possible that the constituents which it receives that
are useful in the organism are in some way transformed and given
back to the circulation. We will recall the fact that the kidneys are
capable of effecting syntheses. Their cells conjugate benzoic acid with
glycocoll. Is there any reason for believing that this is the only syn-
thesis which the kidneys are capable of effecting? We will also mention
the fact that the animal organism is exceedingly economical with its

THE ELIMINATION OF METABOLIC PRODUCTS. 591

supply of phosphoric acid. In increased diuresis the amount of urea
and of common salt in the urine increases considerably, but the amount
of phosphoric acid present remains remarkably constant.

The reaction of the urine depends, as we have already said, upon the
nature of the food. The urine of herbivora is neutral or alkaline, while
that of the carnivora is acid as a rule. The direct connection which
this has with the food may be indicated theoretically by compar-
ing the ash of plants with that of animals. That it is not any par-
ticular difference in the metabolism taking place in cUfferent classes of
animals which causes the different reaction of the urine, may be shown
by feeding vegetables to the carnivora. The urine then becomes neutral
or alkaline. Conversely, herbivora may be forced to become carnivora
by starvation. The animal is then obliged to live upon its own tiissue,
and the urine then has an acid reaction. Alkaline urine, especially in
herbivora, is usually turbid on account of the precipitation of alka-
line earth salts. The urine of a normal man with a mixed diet shows an
acid reaction. The acid reaction is caused by the fact that during meta-
bolism acid products are formed by the combustion of neutral substances
such as albumin and lecithin for example. The sulphur contained in
albumin is largely converted into sulphuric acid, the phosphorus of leci-
thin and of the nucleic acids is oxidized to phosphoric acid. Further-
more, organic acids, as, for example, hippuric acid, uric acid, oxalic acid,
and aromatic oxyacids, are also formed. The organism, moreover, pos-
sesses ways and means for keeping the acidity within certain limits.
For one thing, the acid formed may be neutralized by means of alkali
carbonate; and if there is not enough of this present, then the ammonia
which is set free by the decomposition of proteins comes into play.

It is perhaps well here to make a few general observations concerning
the conception of acidity. An acid may be defined from two stand-
points.' The chemist understands by an acid a substance whose hydro-
gen atom, or atoms, may be replaced by metals. When the metal enters
the molecule the acid character is neutralized. Thus the acidity of a
solution may be estimated by measuring the amount of alkali which is
necessary to replace all of the acid hydrogen. Our discussion of the acidity
of the urine was from this point of view. The physico-chemist, on the
other hand, defines an acid as a chemical compound which when dis-
solved in water is dissociated, yielding positively charged hydrogen atoms
(H"*"). According to the degree of dissociation, we characterize an acid
as strong or weak. A weak acid, for example, is one which at a given
concentration is less dissociated than a strong acid. The difference
between these two points of view maj' be perhaps best illustrated by an

' Cf. R. Hoeber: Hofmeister's Beitrage, 3, 525 (1903;

592 LECTURE XXV.

example. Suppose we have a solution of ^ normal hydrochloric acid,
and one of acetic acid which is also ^ normal. From the purely chemi-
cal standpoint these solutions are of the same strength, because we shall
have to use as much alkali to neutralize a liter of the acetic acid as would
be necessary for a liter of the hydrochloric acid. On the other hand,
from the point of view taken by the physico-chemist, the acidity of the
^5 normal hydrochloric acid is about 40 times as great as that of the g^
normal acetic acid. Thus the hydrochloric acid of the above concentra-
tion is about 97 per cent dissociated, while the acetic acid is only disso-
ciated to an extent of 2.4 per cent. Now in our ordinary chemical
methods we neutralize all of the hydrogen of the acid, because there is
always a fraction of the whole molecule that is dissociated, the value of
the fraction increasing with the dilution; and as fast as some of the
ions are neutralized more of the molecule dissociates, so that eventually
not only the hydrogen ions originally present, what we may designate as
the active hydrogen ions, but also those which were originally undissoci-
ated, the potential hydrogen ions, are neutralized. The physico-chemist
in his determination of the acidity takes into consideration only the
former kind of hydrogen. From his point of view the urine is usually
neutral. There seems to be no definite relations between the acidity
determined by the titration of urine and the so-called ion-acidity. It
is desirable that in all cases both values should l)e known.

In general, not much is known concerning the way in which the dif-
ferent substances proved to 1)e present in urine are combined there. The
analysis of the ash as such teaches us but little. It gives us considerable
information in tracing the course of the non-volatile material, but in this
case the intestinal elimination must not be disregarded. The fact that
the inorganic substances are, at least to some extent, eliminated by the
intestines, complicates our understanding of the general metabolism, and
especially because of the fact that in every case it is impossible to decide
what part of the constituents of the ash of the fjeces is to be re-
garded as unabsorbed material and what part was eliminated from the
intestinal walls after absorption had taken place. The value of mineral
substances for the whole organism and their absolute indispensability
have been repeatedly mentioned, and we are convinced that exact investi-
gations concerning metabolism on as broad a basis as possible, taking
into consideration the inorganic material introduced and that eliminated,
will give us considerable information as to the nature of cell-metabolism.

We must also consider a phenomenon which is frequently met with in
human urine. Fresh urine is usually clear and shows no sediment. After
the urine has stood for some time a sediment often forms, sometimes as
a reddish, crystalline powder, sometimes as a reddish-gray precipitate,
resembling brick-dust. The latter is called Sedimentum lateritium. It

THE ELIMINATION OF METABOLIC PRODUCTS. 593

dissolves completely on heating, and appears again on cooling. On stand-
ing for some time, crystals often appear in the sediment, which will not
dissolve on heating the urine. These crystals are free uric acid, while the
sediment was monosodium urate. The precipitation of the latter is,
partly at least, due to the cooling of the urine, for it is much more soluble
in hot water than in cold. As the crystals form, the acidity of the urine
decreases. The question arises whether the change in the reaction of the
urine has any connection with the deposition of the urate precipitate. We
have at a previous place mentioned the insolubility of uric acid;' it requires
at 18° C, 39,000 parts of water to dissolve one part of the acid. The
values given in the literature are often widely different from the above
value which is taken from the work of His. This is partly explained by
the fact that it is usually disregarded that glass, especially common glass,
contains alkali, which it gives up to water which is in contact with it, and
this affects the analj^sis. His, again, has called attention to the great
tendency of uric acid to form supersaturated solutions. The acid urate
of sodium, also called the mono-urate, is far more soluble in water than
uric acid itself. Now this urate is deposited frequentlj' in urine, and of
other cases free uric acid is found in the .sediment, and, in fact, so much in
it that it is hard for us to believe that it was present as such in the urine.
Camerer ^ compared the solution of uric acid in the urine with the following
experiments. He mixed a saturated solution of acid urate of sodium,
which reacted alkaline toward litmus, with a solution of acid phosphate
of sodium. The mixed solution showed an acid reaction and was perfectly
clear at 37° C. On cooling the mixture, the reaction toward litmus
changed. The solution became alkaline and uric acid was precipitated.
A chemical decomposition had taken place. From the acid phosphate of
sodium (NaHoFOi), and at the cost of the sodium in the monosodium
urate, disodium phosphate (Na2HP0i) had been formed, and uric acid
had been set free; which, on account of its insolubility, was precipitated.
On heating the solution, the reverse process took place, and the reaction
of the urine became acid. In the urine there is always more or less alkali
phosphate present, which may have the same effect as in the above test-
tube experiment. Naturally, according to this explanation, it must be
assumed that the uric acid is originally present as the monosodium salt.
There is no question that part of the uric acid is actually present in this
form; but, on the other hand, it is certain that a part of the uric acid
is present, combined in some other way. This is evident from the fact
that from a simple solution of alkali urate, the whole of the uric acid may
be precipitated by the addition of acid, while this is not the case with
urine. A part of the uric acid remains in solution after the urine has

• Cf. T. Paul: Pharm. Ztg. 1900, also Lecture XIII, p. 298.
' Deut. med. Wochschr. 17, No. 10, p. 356 (1896).

594 LECTURE XXV.

been acidified. Now urea is a good solvent for uric acid. It is an open
question wlietlier we are justified in assuming witli Rudel ' that there is a
chemical combination between the urea and the uric acid in this case,
and it is equally uncertain whether the urea alone affects the solubility of
the uric acid, or whether there are other compounds present in urine which
have the same action.

We have already said that the epithelia of the blood-vessels and of the
uriniferous tubes can only cause the elimination of those substances which
do not belong to the plasma, or which are present in more than the normal
amount. Thus the kidneys, for example, are very sensitive to an increase
in the sugar-content of the blood. Albumin does not pass through the
kidneys when they are acting normally, except when albumins foreign
to the body evade the alimentary canal, and get into the circulation.
It is a well-known fact that under pathological conditions, in diseases of
the kidneys, albumin passes from the blood-capillaries, and enters through
the epithelium of the uriniferous tubes. The presence of albumin in the
urine is a symptom which may arise from a number of different processes.
There is no question that a study of the nature of the eliminated albumin
could be used as a basis for further inquiry. To be sure, in many cases
there is a mere appearance of serum-albumin, but we can well imagine
that in other cases the tissue-cells for some reason or another produce an
albumin, and give it up to the plasma, of a nature which in its entire con-
struction is foreign to the plasma, and that it is accordingly eliminated by
the kidneys. This is not the place to discuss at any length such questions,
which are closely related to the pathology of metabolism.

As we have already mentioned, the organism can under certain condi-
tions eliminate the constituents of the urine through other glands, especially
through the skin. Thus in many cases of uraemia, urea may be secreted
in such quantities by the sweat-glands, that crystals deposit upon the skin.
By ursemia we understand a very serious complex of symptoms, occurring
when the kidneys to a greater or less extent have ceased to exercise their
functions. The organism seeks by every means in its power to get rid of
the constituents of urine which are circulating in the blood. If it does
not succeed in accomplishing this, symptoms appear which are similar to
intoxication. The attempt has often been made to trace the cause of the
disease to some constituent of the urine, and in this connection urea has
been principally considered. On the other hand, there are quite a number
of observations which indicate that the urine itself exerts a poisonous action.
Thus if human urine is injected into the veins of a rabbit, it will produce
acute poisoning, which will result in the death of the animal. The urine
from different animals shows different degrees of poisonous properties.

■ Arch.exper. Path. Pharm. 30, 469 (1892). Cf.T. J. Zerner: Wiener klin. Wochschr.
6, No. 15, p. 272 (1893). A. Ritter: Z. Biol. 35, 155 (1897).

THE ELIMINATION OF METABOLIC PRODUCTS. 595

From the material at hand it is hard to decide as to the significance of the
phenomenon, and there is no indication of what substances in the urine
exert this poisonous action. In the case of urssmia we have no reason
for attributing any one substance as causing the whole complex of
symptoms. It is self-evident that all sorts of different substances may
come into play here, and furthermore it must never be forgotten that an
abnormal composition of the plasma will immediately have an effect upon
all the processes of metabolism in the cells, and result in the production
of incompletely formed products, or of those which are built up in an
unsuitable way. Here, as in all physiological and pathological processes,
an organ should not be considered by itself, but we must trace the
damages which start from it in a continuous line from organ to organ,
from tissue to tissue, and finally from cell to cell.

The animal organism is also normally eliminating substances constantly
through the skin. We find in mammals e.ssentially two kinds of glands
in the skin, the sweat-glands and the sebaceous glands. The former
eliminate a secretion which consists almost entirely of water. The amount
of sweat secreted in the course of a day varies tremendously, and is depend-
ent upon certain conditions, and especially upon the demands for a regu-
lation of the body temperature. In the evaporation of water from the
surface of the body the animal organism finds its most important means
for preventing the body from being overheated. A large amount of heat
is required to transform water from the liquid to the gaseous state. This
causes the body to be cooled. It is interesting to find that the activity of
the sweat-glands is influenced by the central nervous system. A secretion
may be produced directly by nervous stimulation.

The sebaceous glands have a different and more local function. Corre-
sponding to this fact, their secretion has a quite different composition.
In a fresh condition it is an oily, semi-liquid mass, which, on standing in
the air, solidifies on the surface of the skin to a greasy tallow. It contains
fat, albumin and cholesterol. Its most important function is to lubricate
the skin. We will here mention again that a modified sebaceous gland,
the oil-bag of birds, contains octadecyl alcohol CigHsgO,^ and finally that
the mammary glands likewise may be considered as related to the sebaceous
glands.

Rohtnann; Hofmeister's Beitr, 5, 110 (1904).

LECTURE XXVI.
THE EPILATIONS OF THE ORGANS TO ONE ANOTHER.

At the close of the last lecture we referred briefly to the mammary
glands. These glands exercise their function only under certain definite
conditions. The period of lactation does not begin until about the time
the secretion is required by the suckling. Long before the birth, however,
external changes may be noticed showing that the glands, then at rest, are
developing in such a way that they will be able to meet the demands that
are to be laid upon them. We have here an interesting example of the
relation of widely different organs to one another. The function of the
mammary glands is dependent directly upon the generative apparatus of
the female. There must be an intimate connection between the two
organs. Just what this is, we cannot tell. It is generally assumed that
nervous influences cause the coincidence in the development of the preg-
nant uterus and that of the mammary glands. It is perfectly conceivable
that this assumption is correct, although in recent years it has been shown
that many apparently reflex nervous processes may be traced to chemical
reactions. We would recall in this connection the influence of the hydro-
chloric acid from the stomach upon the secretion of the pancreas. The
secretion of the latter is accelerated as soon as the hydrochloric acid enters
the intestine. The simplest assumption was that the hydrochloric acid
irritated the end-apparatus of the nerves in the intestinal membrane, and
thus reflexively stimulated the pancreas into increased activity. It has
been shown, however, that the mucous membrane of the intestine contains
an antecedent, the prosecretin, which is set free by the hydrochloric acid,
and as secretin is carried by the blood-passages into the pancreas. Accord-
ing to this, the alimentary tract, or at least that portion which produces
the prosecretin, falls into line with those organs which are said to produce
internal secretions. It is not right to give a particular position to an organ
shown to produce internal secretions. There is no reason why an organ
which gives up its secretion directly to the blood should be considered as
essentially different from the ordinary glands which send out their secre-
tion through ducts. Numerous observations in physiology and in pathology
compel us to assume that all the organs are in some way related to one
another. We must not be satisfied by merely saying that this connection
is made by means of the nerves. It is far more probable that the separate
cells of the body not only give up the metabolic end-products to the lymph
and blood, but secretions as well. This .view seems in accordance with

596

RELATIONS OF THE ORGANS TO ONE ANOTHER. 597

the entire anatomical construction of the animal tissue and with our
ideas of metabolism.

It would be, in fact, hard to understand why the separate tissues
should be so much differentiated if the e.ssential part of metabolic pro-
cesses consisted merely in enabling the cells already formed to retain their
constituents and in furnishing them merely with sufficient heat units for
the exercise of their functions. If even the digestive process, which
a priori appears so simple, requires such a fullne.ss of chemical processes,
utihzes so many organs, and reacts so sensitively to the different condi-
tions which prevail, we may conclude at once that the cell-metabolism
certainly cannot proceed along altogether simple lineS; but that here
also secretions from certain groups of cells must be of considerable sig-
nificance. We hold that it is not entirely impossible that every indi-
vidual cell of the body takes part in secretory work, and thus has in some
way a favorable effect upon the general metabolism. Perhaps this point
of view may give us some idea of the reason whj' organisms constantly
require a certain amount of albumin. Unquestionably the proteins
occupy a quite different position in metabolism from that taken by the
nitrogen-free foodstuffs. We can well imagine that they are required
chiefly for the formation of secretions. We do not overlook by any means
the fact that the large requirement of albumin is even then only partly
explained unless one is ready to assume that in the formation of the secre-
tions a large number of cells are disintegrated and therefore must be
built up anew.' It seems to us very important to state that there is no
essential difference between the glands with an e.xcretory duct and those
in which there is no duct. It is especially doubtful whether we are justi-
fied in assuming that only cells arranged in the shape of a gland are
active in the formation of secretions. Many facts indicate that the
contrary is true. Moreover, there are many intermediate stages between
glands with ducts and those with none. The mucous membrane of the
intestine secretes intestinal juice, enterokinase externally and secretin
internally. The pancreas secretes externally the digestive juice, and also
probably secretes substances internally which take part in the metabolism
of carbohydrates. Again, the liver undoubtedly has several secretory
functions. On the one hand, it yields the bile which, in its formation
and the method of giving it up, corresponds to an external secretion.
Now we know, for example, that the liver is constantly storing away
sugar and assimilating it as glycogen in order that, at the right moment,
fermentation may cause the reverse process to take place, — i.e. sugar be
given up to the blood. Certainly this is an internal secretion just as
much as the formation of any other substance under the influence of the
cells. To be sure, in this case we know just what this secretion is,

' Cf. Lecture XI, p. 221 el seq.

598 ' LECTURE XXVI.

namely, (i-glucose. The cells of the liver take part in its formation,
inasmuch as they furnish the ferment which hydrolyzes glycogen. We
have already indicated that we must not regard these fermentation pro-
cesses as simple in their natur^. Wherever it has been possible to follow
a fermentation closely, it has been found invariably that it consists of a
whole chain of separate processes. The ferment itself does not origi-
nally exist as such, but in a precursory stage, which is changed to the
active condition by the aid of a product obtained from other cells. We
cannot be wrong in assuming that such relations take part in the breaking
down of glycogen.

We have mentioned these processes particular!}- because they seem
to be the most suitable for demonstrating how the different cells of the
body work together. It is certain that gi-eater clearness would prevail
•with regard to pathological processes if such relations were kept more in
the foreground in each instance and the diseased organ itself, not so much
regarded as representing the whole " case." If we study all of the com-
plicated processes which take part in a single fermentation from the
beginning to the end, we shall realize at how many different places dis-
turbances in the normal course of metabolism may arise.

Let us now return to the functions of the mammary glands. These
may very likely be excited into lactation by means of a secretion pro-
duced by the pregnant uterus, or its accessories. The transformations
taking place in the dormant mammary gland from the beginning of its
preparatory period to the time when it enters into the full exercise of its
functions, are profound. There is an extensive formation of new cells.
The cells of these glands, which, like all other cells of the body, receive
their nutriment from the blood, suddenly make new demands upon
it. They abstract a great deal of material from the blood, which they
transform considerably. They form casein from the albumins of serum,
and lactose from d-glucose. Again the salts are removed in definite
amounts and quite independently of the ratio in which they are present
in the blood. We have previously gone into these details. How the
cells of the mammary glands accomplish these changes is not known.
We do not know of any intermediate stages between the serum-albumins
and casein, nor between grape-sugar and milk-sugar. We can merely
imagine that maltose is formed in the production of the latter. Before
much was known concerning the composition of the various different
proteins, the transformation of serum-albumin into casein did not appear
to be a very complicated process. To-day we are already compelled to
assume that before casein can be formed, the serum-albumin must be
decomposed to a considerable extent, after which a synthesis is effected.
The cells of the mammary gland do not in principle assume any extraor-
dinary position. Their chemism is merely an individual and a specific

RELATIONS OF THE ORGANS TO ONE ANOTHER. 599

one, and consequently the products which they form are typical, just
the same as the sahvary glands yield one specific secretion and the pan-
creas another. There are not any essential differences between these
processes. We should be making a sad mistake if we were to consider
the function of the cells of the mammary glands by itself. We are
able to understand it only when we trace its phases from a general stand-
point. The cells of the mammary glands take up from the blood, or
more directly from the lymph, certain substances which evidently must
be transformed completely, and to a certain extent assimilated, in order
to form the product which it will subsequently give up. Recent inves-
tigations indicate that even the actual process of secretion is not different
from that of other glands, for here also, after the secretion has been given
up by the cells, a residue of protoplasm and nucleus remains, and a new
formation of the same secretion again ensues.

It is not alone the mammary glands that are dependent upon the sexual
apparatus. More and more we become cognizant of the fact that the
different parts of the latter stand in a number of different relations to
other organs, though we are not able to discover the nature of the active
principle any more definitely. We know at present merely of isolated
facts which we cannot explain satisfactorily. The different female sexual
organs are, in the first place, related to one another. As an example of
this, we need only cite the influence of the ovaries, when they are exerting
their normal function, upon the uterus, and especially upon the mucous
membrane of the uterus at the time of menstruation. Here again we
frequently find this attributed to nervous excitement, although there is
no definite proof that such is the case. The experiments of Halban '
have shown that the ovaries can exercise their function when there is no
connection with the nervous system. He showed that if he extirpated
the ovaries from young guinea pigs and inserted them at another part of
the body, the development of the external genitals took place exactly as if
the ovaries had remained in their original position. On the other hand,
in immature animals in which the ovaries had been completely removed
from the body, there was a halt in the development of the external organs
of generation. It is also well known that when the function of the
ovaries ceases, whether due to their ablation in a mature condition, or to
the fact that they have reached the end of the period of their activity,
changes take place in the uterus corresponding to a retrogression.

Similarly the male organs of generation stand in relation to one another.
This is already evident from the waj' the cells of the testes work together
with those of the prostate gland, although this may be explained as a
result of a common stimulation. There are observations according to
which the prostate atrophies after the removal of the testes.

' Monatschr. f. Geburtshilfe u. Gyniikol, 12, 496 (1900).

600 LECTURE XXVI.

The relations of the sexual organs to the entire organism "are very inter-
esting. By numerous experiments on men and animals, it has become
well recognized that extirpation, the so-called castration, before sexual
ripening has taken place, tends to prevent the formation of the secondary
se.xual character. This is well illustrated in cocks, which, as we all know,
when fully developed sexually may be recognized by their wattles and
combs. These remain undeveloped, or at least are but scanty, if the testes
are removed before the sexual development is complete. It is interesting
also to find that a secondary sexual character develops if the extirpated
testes are transplanted after their removal from the fowl.'

One of the most prominent results of the removal of the sexual glands
is an abnormal growth of the bones. In castrates it is frequently found
that especially the tibia and the femur are prolonged. The cause of this
has been traced to a faulty ossification of the epiphysis cartilage such that
there is no limit placed upon the growth of the bone. Apparently castra-
tion afTects the general metabolism. The great tendency of castrates
towards obesity is well known. It has never been positively established
whether this results primarilj' from the loss of the sexual glands or whether
it is a secondary effect.

Although there is undoubtedly a connection between the sexual organs
and the other organs of the body, still at present we are unable to identify
any definite product of their secretion as the active principle. We know
of glands, however, which are ductless, but do not give rise to such
secretions. We refer to the suprarenal bodies and the thyroid gland.

The extirpation of the two suprarenal capsules has been made by Brown-
Sequard,^ whom we have to thank for many investigations in this line of
research. He found that their removal caused death in a short time. He
was able to keep the animal alive, if a part of one of the capsules was allowed
to remain. The animal soon lost in weight and showed a peculiar behavior.
It was lazy, and if compelled to work soon became very tired. One
of the pronounced effects was that the blood-pressure fell immediately
after the operation. The fact that the blood from such animals had toxic
properties, and when injected into normal animals led to similar symp-
toms, as in the animal which had undergone the operation, gave rise
to the assumption that the suprarenal capsules served to destroy those
products formed by metabolism which are injurious. According to this
view, the suprarenal bodies serve merely as a means of protection. There
is no conclusive proof of the correctness of this assumption. It is clear
that the extirpation of these capsules may influence metabolism in such
a way that when they are removed from the body some product circu-
lates through the body in an abnormal condition, and the toxic properties

' Foges: Pfliiger's Arch. 93, 39 (1903).

' Corapt. rend. 43, 422 and 542 (185G); ibid. 45, 1036 (1857).

RELATIONS OF THE ORGANS TO ONE ANOTHER. 601

of the blood may be due to this fact. It is perfectly unjustifiable to
assume that substances appearing after the extirpation of an organ are
in any way to be regarded as normal products, and that they are simply
not removed because a certain organ is missing. It is possible that such
an assumption does represent the truth, but at the same time it is equally
probable that the injurious substances are formed because the organ is
not present.

It has only quite recently been shown that the suprarenal botlies do
actually give up to the blood a specific substance. It has been found
possible to isolate this substance and crystallize it. ■■ Oliver and Schafer' had
observed that extracts of the suprarenal capsules when injected into the
veins gave rise to a marked increase in the blood-pressure. These investi-
gators traced this increase of pressure to a strong contraction of the blood-
vessels, and also ,to the fact that the suprarenal extract had an action upon
the heart. Long before this, in 1856, Vulpian ^ had his hands upon this
active principle. He found that the suprarenal bodies contained a sub-
stance, the so-called chromogen substance, which turned dark on exposure
to the air, and gave with ferric chloride solution a green coloration. It
is only recently, however, that it was found possible to prepare this active
principle in a pure state. Its discovery was not alone of physiological
interest, but at the same time a remedy was added to our store, which
met with a favorable reception such as is but seldom accorded to a new
preparation. It is used especially to prevent bleeding in surgical opera-
tions. The composition of the substance corresponds to the formula

CgHisNOg.^

According to E. Friedmann * it has the following structure:

H
C
■^ \
HOC C-CH(OH) .CH2.NH.CH3

I !l
HOC CH

C
H

' J. Physiol. 16 (1894); 17, IX (1894-95).

2 Compt. rend. 43, 60.3 (1856).

3 Cf. von Fiirth: Z. physiol. Chem. 24, 142 (1898); 26, 15 (1898-99); 29, 105 (1900);
Hofmeister's Beitrage, 1, 243 (1901); Sitzber. kais. Akad. Wissensch. in Wien. Math.-
natur. Klasse 112, Abt. 3 (March 5, 1903). Abel and Crawford: Johns Hopkins Hosp.
Bull., No. 76 (1897). J. Abel: *!'(/. No. 90-91 (1898); Am. J. Physiol. March, 1899;
Z. physiol. Chem. 28, 318 (1899); Johns Hopkins, Bull., No. 120 (March, 1901); No.
128 (Nov. 1901); No. 1.30, 131 (Feb.-March, 1902); Am. J. Physiol. 8, 2 (1903);
Ber. 36, 1839 (1903). J. Takamine: Am. J. Pharm. 73 (1901). H. Pauly: Ber. 36,
2944 (1903). Abderhalden and Bergell: ibid. 37, 2022 (1904).

« Hofmeister's Beitrage, 8, 94 and 118 (1906).

602 LECTURE XXVI.

Several names have been assigned to the substance, but that of adrenalin
seems most suitable. By fusing it with caustic potash, protocatechuic
acid is obtained. By the action of mineral acids, methylamine is split off.
Among the other cleavage-products, pyrrole, methyl indole, and pyridine
have also been observed. We are not much better informed concerning
the formation of adrenalin than we are concerning its constitution. We
do not know its source, although it is possibly derived from the proteins
and their disintegration products.

As little as 0.0013 milligram of adrenalin will cause a noticeable increase
of the blood-pressure when introduced into the circulation. At the same
time it strengthens the heart-action. The peripheral vessels become
strongly contracted. Mucous membranes appear nearly white on account
of the almost complete absence of blood in them. Adrenalin also acts
upon the dilatator pupillw, retractor memhrana nictitans, and the smooth
muscles of the eyelids.' These muscles contract when under its influence.
The movements of the stomach, the gall-bladder, and the urinary-bladder
on the other hand are possibly restricted.^

How shall we imagine that the suprarenal capsules act in the economy
of the animal organism? Evidently they are constantly giving up adrena-
lin to the blood either as adrenalin or perhaps in some kind of combination
such that all the organs innervated by the Sijm pathetic us are affected
by it. It is certainly interesting in this connection that neither their
development nor anatomical structure indicates that these capsules are
homogeneous organs. There are two distinct parts of the capsule, one of
which is derived from a collection of mesenchymatous cells in the vicinity
of the inferior vena cava and gives rise to the cortex, while the other origi-
nates from the al:)dominal sympathetic ganglia and forms the medulla.
There is considerable evidence which points to the fact that adrenalin is
produced by the cells of the medulla. We have, therefore, in a wide
sense an innervation process before us which is brought about by the aid
of a chemical product. It is certainly not without significance that the
Sympatheticus, or rather an organ derived from it, produces a substance
which acts upon it and the organs innervated by it. At present there is
nothing definitely known as to whether the sympathetic nerve influences
this internal secretion, but there is some evidence which indicates that
such is the case.

We have already mentioned that there has been ascribed to the supra-
renal bodies an action in combating poisons. Although we do not doubt
in any way that these organs may have other functions than that of pro-
ducing an internal secretion, still there is nothing positively known as to

' M. Lewandowski: Zentr. Physiol. 12, .599 (1S98).
' Boruttau: Pfliiger's Arch. 78, 97 (1S99).

RELATIONS OF THE ORGANS TO ONE ANOTHER. 603

the nature of such functions. It may be mentioned that large doses of
adrenalin produce glucosuria. It results from a glucohemia the cause of
which is still obscure.

We may also mention that a disease is known to pathology which is
related to the suprarenal glands. It is named after its discoverer, being
known as Addison's disease.' The most important symptoms are as
follows: There is an external pigmentation upon the skin. The mucous
membrane also shows dark-colored patches, and there is a marked antemia
and extreme asthenia. Great muscular weakness characterizes the whole
course of the disease. It is shown not alone in the inability of the muscles
to perform work, but by the fact that they quickly become tired. We
remember that these same symptoms were observed in animals with
ablated suprarenals. All of the other symptoms in Addison's disease are
of a secondary nature, and are due especially to the marked anaemia. A
post-mortem examination shows that the suprarenal bodies are more or
less destroyed by tumors, and usually as a result of tuberculosis. We must
not fail to mention, however, that cases of the disease are known, in which
the suprarenal capsules remain anatomically " normal." We have repeat-
edly laid stress upon the fact that the anatomical appearance of an organ
does not always indicate whether the organ is exerting its normal function
or not. Only when it has been found possible to show that the complex of
symptoms of Morbus Addisonii can exist without there being any change
in the junctions of the suprarenal bodies, shall we be justified in questioning
the connection of the above-mentioned disease with the suprarenal bodies.
There has been a great deal of discussion as to the origin and significance
of the bronze-colored patches upon the skin. The nature of the deposited
pigment is not known. It is possible that it has some relation to the
formation of the secretion by the suprarenals, and that it may perhaps
represent an antecedent of adrenalin, which is deposited because in that
condition it is of no benefit to the system. On the other hand, we
must guard against putting the secretion of adrenalin too much in the
foreground, simply because it is the only secretion of the glands of which
we now know. It is pretty certain that the suprarenals have other func-
tions, and it is perfectly possible that when these functions disappear the
results are more serious than when the known secretion alone is wanting.
Our knowledge is still far too limited for us to attempt to discuss the
relations of the individual symptoms of the disease to the suprarenal
bodies. Above all, the cause of the extreme lassitude of the patients
remains unexplained.

Another very important organ is the thyroid fjland. Its functions were
investigated constantly for years, without its being possible to ascertain

' T. Addison : On the Constitutional and Local Effects of Disease of the Suprarenal
Capsules. London, 1855.

604 LECTURE XXVI.

■what they were, and the attempts to isolate the active principle contained
in the organ were fruitless. Here again we have oljservations from the
fields of physiology and of pathology at our disposal. We shall begin
with the latter. The thyroid gland frequently shows signs of degenera-
tion, and this apparently takes place in a definite manner, and is most
common in definite localities. These are the so-called goitre regions; i.e.,
regions in which there is frequently a cystic deformity producing an
enlargement of the thyroid. The nature of the soil, and especially of
the drinking-water, has been assumed to be the cause of the disease, with-
out 'any one being able to show this conclusively. Frequently other
severe derangements accompany the deformity of the gland. The mental
development of those afflicted with the disease is slight. Such individuals
are known as cretins. There is a derangement of their entire metabolism.
Scholz ' in particular, who studied a case very carefully, showed this to be
true of the metabohsm of albumin and salts. We are justified in assum-
ing, as we shall soon see, that the thyroid gland has not ceased entirely
to exercise its function. It seems highly probable that we shall realize
more closely the part taken by the thyroid gland in metabolism, if we
assume that it has different functions. We can well imagine that in
cretinism it is possible for the thyroid to retain certain of its functions
while others are missing.

An idea as to the complete function of the thyroid gland is probably to
be obtained best by studj^ing an organism after the complete ablation of
the organ. The operation has been performed upon man as well as upon
animals; in the first case, however, only at a time when we were not yet
ready to study the whole consequence of the interference. To-day it is
well recognized that the organ is quite essential, so that care is taken not
to remove it completely. We may say, in this connection, that a small
portion of the gland left in the body is usually sufficient for the retaining
of all the functions of the organ. This fact should be well borne in mind,
for it gives us the key to the cause of many contradictions which are to be
found in the literature. The thyroid gland itself is an unpaired organ.
Anatomically it is usually homogeneous. Embryologically it arises from
clefts or bronchial arches at the lower part of the epithelium of the
pharynx. Often, however, we find near the main gland isolated fragments
of similar material in the surrounding tis.sue, and frequently these are
quite far from the main gland. In the ablation of the latter, they may
assume its functions, so that the typical results of the operation are not
felt. Entirely distinct from the thyroid gland are the parathyroid glands.
They are paired, and originate from the last pair of clefts of the pharyn-
geal epithehum. We shall find, later on, that their position relatively to

' Z. exper. Path. u. Therap. 2, 271 (1905).

RELATIONS OF THE ORGANS TO ONE ANOTHER. 605

the main thyroid gland varies in different animals. Their significance has
only very recently been realized.

If the thyroid gland is completely removed from the body, or its functions
fail for any reason, peculiar changes result. They were first observed
and described by William Gull ' in 1874. The most prominent symptom
is a thickening of the skin. It appears, on account of the increased
amount of mucin in the subcutaneous connective tissue, as an edematous
swelling. For this reason Ord - gave to the disease the name, 7nyxedema.
Subsequently the swelling goes down, and the skin then appears more
atrophied. The secretion of the glands in the skin ceases, and the latter
becomes hard and dry. Metabolism is disturbed, and so is the temperature
of the body, and the mechanism for regulating the body-temperature.
The most striking disturbances are those of the muscular and nervous
systems. They are of various kinds. Sometimes there is evidence of
increased sensitiveness, while in other cases the change is entirely in the
other direction. The various effects, which also change as the disease
progresses, may be due to the more or less complete failure of the functions
of the gland.

J. L. Reverdin, A. Reverdin,^ and afterwards Theodor Kocher,^ had
considerable opportunity to study the effects of the total extirpation of
the organ in man. They found on the whole the same symptoms as in
myxedema. Kocher embraced the whole complex of symptoms under
the name of Cachexia strumipriva. It is not a simple disease. In general
the same characteristics are manifest. In individuals which have not
attained full growth, extirpation of the organ causes a tardy develop-
ment of the length of the bones. We find here reminiscences of cretinism.
It is specially noteworthy that individuals quickly lose the ability to
reason. Finally idiocy may result.

After J. L. Reverdin had published his first results, physiologists recalled
the experiments of Moritz Schiff ^ made in 1859, with regard to the total
extirpation of the thyroid gland in animals. Schiff showed that dogs
did not long survive the operation. They died within from 4 to 27
days. There is to-day no doubt prevailing as to the correctness of
his observations, although the cause of the different behavior of vari-
ous classes of animals has been much disputed. With dogs and cats
death results quickly and usually in a convulsive attack (tetany). Muscu-

' Trans. Clin. Soc. London, 1874.

' On Myxoedema. Medic-chirurgical Transactions, Second Series, 43, 57 (1878).

' J. L. Reverdin: Revue m^dicale de la Suisse romande, 2ierae ann^e, 539 (1882),
and 3ieme ann^e, p. 47 (1883). J. L. Reverdin and Aug. Reverdin: ibid. 3ieme annee,
No. 4, pp. 169, 233, 309, and 686 (1883).

* Arch. Clin. Chir. 29, 254 (1883).

5 Arch, exper. Path. Pharm. 18, 25 (1884).

606 LECTURE XXVI.

lar tremors first appear, which gradually pass into clonic spasms, finally-
resulting in tetanus. The muscular tremors are not of peripheral origin,
for they disappear on section of the peripheral nerves. Apparently the
thyroid gland in some way influences the higher nerve centers. It is
evident, however, that the lower nerve centers are also affected, for the
tremors continue aftei' the removal of the cortical brain area concerned
with the movement of the part. In the case of herbivora, the ruminants,
rodents, and monkeys, tetany does not as a rule take place. Instead,
cachexia becomes a prominent symptom. This contrast of symptoms in
the two classes of animals, which was made more puzzling by reason of
the fact that with the herbivora sometimes tetany appears and sometimes
does not, has recentlj' been offered an explanation. We have already
mentioned the presence of the parathyroid glands. In the carnivora
these are included in the main gland, whereas in the herbivora they are
separated from it. For this reason the parathyroid glands are always
removed from carnivora in cases of complete ablation of the thyroid
gland, whereas in the herbivora this is rarely the case. In fact, it has
usually been found that tetany in herbivora results when these parathy-
roid glands are removed.' According to this discovery, it would seem
that the parathyroid glands and the main gland have different functions.
It seems highly desirable that clinical observations should receive renewed
study with regard to this point.

It might be objected with regard to the experiments in the ablation of
the thyroid gland that the operation itself may be such a severe one that
other injuries can produce .some, at least, of the observed symptom com-
plex. This objection, however, has been successfully refuted by means
of a great many experiments. In the first place, the entire operation
may be performed, except that the gland is allowed to remain in place,
without any of the symptoms occurring. Again, if a part of the organ
is allowed to remain in the body, the symptoms do not appear; and,
finally, if a part of the organ is transplanted to another part of the body,
the whole operation may then be carried out without fatal results. Such
experiments were performed by Schiff and have been repeated by Eisels-
berg ^ in a particularly convincing manner. The latter extirpated half of
the thyroid gland from a cat and grafted it in the wound between the
abdominal fascia and the peritoneum. Then, after this had been accom-
plished, the other half of the organ was carefully removed. The animal
was kept under observation for two months without its showing any

' Cf. E. Gley: Compt. rend. soc. biol. Paris (9), 841 (1891). Vassale et Generali:
Arch. ital. biol. 25, 459 (1896); 26, 61 (1896). Biedl: Innere Sekretion, Berlin, 1904.
MacCallum: Zentr. allg. Path. u. path. Anat. 16, No. 10 (1905).

' A. Freiherr von Eiselsberg, Wiener klin. Wochschr. 5, 81 (1892).

RELATIONS OF THE ORGANS TO ONE ANOTHER. 607

indication of a cessation in the functions of the gland. Then the grafted
piece of thyroid, which showed normal gland-tissue, was removed. The
very next day tetany resulted, and the animal died at the end of the
third day.

It is also important to learn that it is possible to prevent the severe
disturbances resulting from the ablation of the organ, by injecting thyroid
juice into a vein or under the skin, and even by feeding it, or raw thyroid,
directly. In fact, it is even possible to improve the condition of the
patient who has already begun to feel the effects of the operation. It is
seldom that a therapeutic conception can be demonstrated so clearly
and so strikingly as in the treatment of Cachexia strumipriva, and true
myxedema, by means of thyroid preparations. The swelling of the skin
goes down, and the mental faculties are noticeably improved. In a short
time the habits of the patient are so changed that almost nothing remains
to indicate the original severe disease.

As soon as the action of the thyroid gland became understood, attempts
were made to isolate the active principle; but, up to the present time,
such attempts have been in vain. It was indeed believed, for a
short time, that the goal had been reached when E. Baumann,' after
making the important discovery that the thyroid glands of many animals
contain iodine, succeeded in isolating an amorphous substance, the so-called
iodo-thyrin (or thyro-iodine). To-day the relation of this substance to
the organ is still very vague. It contains phosphorus and about nine
per cent of iodine. Now there Ls no doubt that iodine itself has an
effect upon the thyroid gland, and, in fact, even when it is administered,
not in the form of an organic compound, but as free iodine. Often an
existing swelling of the gland subsides. It is still an open question how
the iodine acts, but we are aware that it has a favorable effect upon
various other filtration processes and facilitates, for example, the absorp-
tion of exudates. To be sure, iodine apparently ha.s a quite specific
action upon the thyroid gland. The fact, however, that iodine may be
absent from a normal organ, makes it seem doubtful whether one is on
the right road in assuming that iodo-thyrin is the active principle of the
gland. It seems that possibly too much attention has been paid to the
iodine constituent. It also must not be forgotten that we have no
guarantee for assuming that iodo-thyrin is itself a simple substance. It
is more probably a mixture of several different products. At all events,
it is certain that the gland itself is more active than iodo-thyrin, and so
are all the preparations which contain as many glandular constituents
as po.ssible.

' E. Baumann: Z. physiol. Chem. 21, .319 (1895-96). Baumann and Roos: ibid.
21, 481 (1895-96). E. Baumann: ibid. 22, 1 (1896-97). E. Roos: ibU. 22, 10
(1896-97); 25, 1 and 242 (1898). A. Oswald: ibid. 23, 265 (1897).

608 LECTURE XXVI.

We are still very far from being able to trace the functions of the thyroid
gland to definite chemical processes. We merely understand the effect
of its extirpation, and know, furthermore, that it stands in some relation
to the sexual organs. It has been observed that at the time of menstru-
ation, during pregnancy and lactation, there is frequently a swelling of the
gland. To be sure, processes may be involved here which have nothing
whatever to do with the cell-functions of this organ, but may be caused by
vascular influences. Again, the observation that in cretins the sexual
organs frequently remain undeveloped, does not necessarily prove that
there is a direct relation between the thyroid gland and the sexual organs.
It is certainly not remarkable that in the general metabolic disturbance
even the sexual organs, which as a rule require a constant supply of mate-
rial as they in a certain sense are constantly growing, will likewise suffer
to a marked extent. At present it is impo.ssible for us to distinguish here
between primary and secondary phenomena.

That the thyroid gland yields a secretion, cannot be doubted. This is
evident alone from its histological structure. Apparently the follicular
cells produce the specific secretion. It then passes through openings in
the follicular walls into the lymph, and is then given up to the blood.'
We will merely mention the fact that Oswald isolated from the secretion
of the follicles (the so-called colloid) two proteins, the so-called thyreo-
glohulin and a nucleoproteid. The former alone contains iodine.^

Our limited knowledge concerning the chemical processes taking place
in the thyroid gland makes it impossible for us to in any way give a precise
description of the nature of the function of this very important organ.
Everything is hypothetical. According to the autotoxication theory, it is
the purpose of the thyroid gland to remove, or render innocuous, one or
more toxic substances which would otherwise accumulate in the blood.
There is no gi-ound for this assumption, but it is perfectly conceivable that
the organ can secrete substances which are capable of combining with other
products. It is possible that the iodine content of the thyroid gland
serves such a purpose and perhaps incUcates the presence of easily replace-
able substances. It is, however, also very probable that the thyroid
gland secretes substances that take part in the general metabolism and
regulate chiefly the transformations which albumin undergoes. It is not
at all difficult to formulate assumptions in this direction, particularly after
repeatedly meeting with facts which show that in order to accomplish
fermentation, a number of different body-cells act together. One cell
yields an activator of the ferment, and another the ferment itself. It is
perfectly conceival^le that the thyroid gland is active in this sense, and

' Htirthle: Pfliiger's Arch. 56, 1 (1894).

2 Z. physiol. Chem. 27, 14 (1899). Hofmeister's Beitrage, 2, 545 (1902). Z. physiol.
Chem. 32, 123 (1901).

RELATIONS OF THE ORGANS TO ONE ANOTHER. 609

that it perhaps secretes a kinase which is for the good of all the boch'-cells.
But all this is speculation, and drawing inferences from analogy without
any real foundation. We must not fail to repeat here that the functions
of the thyroid gland are not all of the same kind. It may also serve to
start certain processes.

We must now consider an action of the thyroid gland bearing a certain
analogy to a disease, which, to a certain extent, is the exact opposite to
Cachexia strumipriva. We refer to Basedow's disease. If too much thyroid
gland is administered, there results an abnormal destruction of albumin.
The elimination of nitrogen in the urine increases considerably. Further-
more, there is an apparent intoxication, with increased pulse frequency,
polyphagia, polydipsia and polyuria. In Basedow's disease similar symp-
toms appear, especially the increased destruction of albumin. This
disease has been traced to an increased activity of the follicular epi-
thelium of the thyroid gland. Quite recently the parathyroids have
also been held to be partly responsible. There are many observations
which indicate such a connection,' but it has been by no means positively
established.

The hypophysis, or pituitary gland, is always mentioned in connection
with the thyroid. It is a compound organ. The anterior lobe is glandular
and resembles somewhat the thyroid body, while the posterior portion
consists chiefly of fibrous tissue. Between the two lobes there is a
hollow space rich in vessels and lined with ciliated epithelium. The
function of this body, which was once considered to be a rudimentary
organ, is still unknown to us. In cases of myxedema there has frequently
been hypertrophy of the pituitary gland, while extirpation of the thyroid
tends to produce the same effect. In cases of hypertrophy and enlarge-
ment of the pituitary body peculiar symptoms often develop, especially
an abnormal growth of the bones at the end of the extreinities, the
phalanges of the fingers and toes, although the softer parts, as the hands,
feet, lips, tongue, and nose, are also affected. It is quite natural to
compare this increased development with the retarded growth which takes
place after the cessation of the functions of the thyroid gland. Yet we do
not positively know that there is a direct connection with this disease,
known as acromegaly, and the changes in the functions of the pituitary
gland. Experiments carried out to determine the functions of this organ,
by studying the effects of its removal, have not led to conclusive results.
Pituitary e.xtracts have also been administered and found to cause an
increased elimination of nitrogen.^ We are not justified in drawing anv
conclusion from this observation as to any existing analogy with the thyroid

' L. Humphry: The Parathyroid Glands in Grave's Disease. Lancet, 11 (1905).

2 T. Malcolm: J. Physiol. 30, 270 (1904). Thompson and Johnston: ibid, 33, 189 (1905).

610 LECTURE XXVI.

gland. The increased elimination of nitrogen may be caused in many
ways. We shall not be in a position to decide such questions until it has
been found possible to isolate the active principles from each of the
organs.

We now turn to two other organs for which a specific function has
been suggested. These are the spleen and the thymus. The latter is a
temporary organ, being a true organ in the case of man only during infancy.
After the child is two or three years old it no longer develops, but slowly
and steadily atrophies, and has nearly disappeared by the fifteenth year,
though traces of it remain in old age. It can be completely extirpated
without causing death. It is, therefore, not to be classed with the
organs that are essential to life. Its ablation is said to result in disturb-
ances in the general health and in metabolism; but from the data at hand,
it is not possible for us to obtain a very clear conception of the func-
tions of the thymus gland. Similarly, its anatomical construction is not
instructive.^

We are almost as much at sea concerning the significance of the spleen
in the economy of the animal organism. All sorts of different functions
have been ascribed to it. It has been said to influence the activity of the
pancreas, an assumption which is not well founded. It has also been
assumed that it plays a part in the production and destruction of the
red corpuscles, and furthermore that it is able to remove and store up waste
material from the blood and lymph. This much is certain, however: the
spleen can be extirpated completely without any severe consequences.
It would be of course unjustifiable to conclude from this that the spleen is
an organ of subordinate importance. Everything depends upon the con-
ditions under which the functions of an organ are tested. It is perfectly
possible that under certain conditions the absence of the spleen might
make itself felt. It may be mentioned, in this connection, that great
importance has been ascribed to the spleen in combating disease germs.
In the case of infections, the spleen sends out a great number of leucocytes.
On the other hand, there are certain indications of the fact that the spleen
on account of its anatomical construction is called upon to regulate the
composition of the blood so that the cellular elements are kept in such a
condition that they are capable of exercising their functions. Abnormal
red and white blood-corpuscles are held back and destroyed. It is possible
that the proteolytic ferment found in spleen, which has an action upon
fibrin, may be active in the breaking down of these discarded elements.
On the other hand, the high iron content of the spleen, to which our
attention has been called repeatedly, is not necessarily to be regarded as

' A. Friedleben: Die Physiologie der Thymusdriise (1858). Cf. J. Aug. Hammar:
Pfliiger's Arch. 110, 337 (1905). Rudolf Fischl: Z. exper. Path. Therap. 1, 388 (1904).

RELATIONS OF THE ORGANS TO ONE ANOTHER. 611

absolute proof of the destruction of blood-corpuscles, for it collects cellular
decomposition products from other organs.' Many observations make it
seem probable that the spleen is related in some way to bone-marrow.
These two substances can mutually aid one another.

In discussing the individual organs we have lost sight of the chief con-
stituents of the entire organism, namely the muscles, nerves, and the con-
nective-tissue group. Of the latter, especially bone, cartilage, and true
connective-tissue, we scarcely assume any intimate relations with the other
organs, although undoubtedly such relations do exist. We are accustomed to
consider them merely as mechanically-acting structures, and for this reason
but little attempt has been made to ascertain what metabolic changes take
place within this group of tissues. As a rule, investigators have been con-
tent with the study of theif chemical composition without attempting to
determine positively the extent to which they take part in the general metab-
olism. Now this connective-tissue group of substances takes part quite
extensively in building up the organism, and its functions are not always
the same. This is particularly true of connective-tissue itself, which,
according to its histological construction, is related to different groups.
For one thing it forms the fundamental support of the body-cells, which,
to a certain extent, are eml^edded in it. From it the finer and coarser
network, in which the lymph passes until it reaches the individual cells,
is formed. It is very questionable whether one is justified in a.ssuming
that the cells of connective-tissue play a passive role here, or whether it is
not more probable that they take active participation in the exchange of
material between the blood and the lymph, and between the latter and
the remaining cells of the body. Its adjustment to purely mechanical
requirements is evident from the construction of the individual tissue;
for example, in the segregation of elastic fibers. A particularly differen-
tiated tissue, and one which also belongs in this group, is the fatty tissue;
the importance of which we have already considered.

As regards the physiological functions of cartilage, but little is known
beyond its purely mechanical properties. It is, however, not to be doubted
that in it, as well as in body tissue, there is a constant occurrence of metabo-
lism. Growth never really ceases, for new cells are constantly being
formed. Numerous observations prove to us the dependence of the
metabolism in these organs upon the requirements placed upon them.
Their development ceases if for any reason there are no demands placed
upon them, and in such cases they retrogress even if they are already
well developed. The influence of the thyroid gland and other organs upon
growth, we have already mentioned. Very active metabolic processes
unquestionaljly take place in cartilage and bony tissue of the growing

' Blumenreich andjacoby: Z. Hygiene u. Infectionskrankh.,29, 419 (1S9S). G. Javvein:
Virchow's Arch. 161, 401 (1900).

612 LECTURE XXVI.

■ organism. These two tissues are intimately related to one another. The
latter can act for the former within certain limits. We find here quite
extensive assimilation processes, and at the same time there is a consid-
erable wearing away. It is seldom that we obtain such a deep insight into
the transformations of tissue as in the case of the new formation of bones.
To be sure our knowledge in this direction is almost wholly morphological.
There has been but little attempt to study this interesting process from a
physico-chemical standpoint. Even the fully-developed bone retains,
especially as regards its periosteum and medulla, its embryonal charac-
ter. From these, new bone material may be formed continually. Thus
fractures are healed. Even under normal conditions, however, there is
continuously taking place a fusion and new formation of bone-substance.
Occasionally we notice the appearance of peculiar cells, the so-called
osteoclasts, which cause the dissolution of bony tissue at the place where
they appear, while on the other hand we meet with the so-called perforating
fibers, which also destroy bone.s. In pathological conditions often the
real disappearance of bony tissue is preceded by decalcification as a primary
process. It is interesting to trace the course of all these processes, in
order to ascertain how, in each separate case, the breaking down of the
bony substance is effected, what agents are active in the process, and why
it is necessary that this fusing together of bone and new formation of such
tissue are constantly taking place. We must for the present allow all these
and similar questions to remain unanswered. We mention these relations
especially because we are indisputably justified in assuming that if in a
ti.ssue to which we are accustomed to assign a very specific function, and
in which we would scarcely expect a -priori that important metabolic
proce.sses would take place, there is a constant exchange of material, so
much more will this be true of all the other tissues which are intimately
connected with active metalwlism and upon which great demands are
placed, and that they will likewise participate in an active interchange of
cell-material.

Such an assumption appears to be particularly justifiable for those
organs whose activity, within certain limits, is a continuous one, as is true
especially of muscular and nervous tissue. The latter, as we well know,
is never at rest. Impulses are constantly passing along the nerve fibers,
partly towards the central nervous system, and partly from this to the
peripheral organs. The nerves are very intimately connected with the mus-
cular tissue. This is evident even from the entire development of the
organs, for they early enter into relations with one another. We know
also that if the innervation ceases, retrogression soon results, bringing on
atrophy, and, in fact, this is evidently due in part to the inactivity of the
muscles. Unquestionably, the nerves have to some extent a direct influ-
ence upon the metaboHc processes in the cells of the muscles themselves,

RELATIONS OF THE ORGANS TO ONE ANOTHER. 613

perhaps in the same way as we found in the formation and breaking down
of glycogen in the Hver that there was an indeterminable dependence upon
the nervous system. Unfortunately, in comparison with the countless
observations of pure physiology concerning the functions of the entire
nervous system, we have nothing from the physiological-chemical stand-
point. In fact, aside from the knowledge of a few constituents of the
nerve substance, we know very little indeed concerning the metabolism
taking place in nervous tissue. We will give here the results of the analysis
of the gray and white substances of the brain.'

White Sub-
stance.

Gray Sub-
stance,

Water

695.35
304.65
25,11
50.02
18.19
26.96
2.94
18.93
5.23

769 97

230 03

10 80

Insoluble albumin and connective-tissue

60.79
6 30

Cholesterol, combined

17.51
1 99

10 43

5 62

The most striking value — and this holds also for the composition of the
peripheral nervous system — is the high phosphorus content of nervous
tissue. Phosphorus is evidently found in very different states of combina-
tion, at one time as nuclein, again in the form of a substance which is
known as protagon,^ and at other times as lecithin. The latter occurs
partly free, and partly results from the hydrolysis of different products,
which have been isolated from nervous tissue, and have been designated
by different names; thus in the decomposition of protagon among the fatty
acids and other cleavage-products, the so-called cerebrin has been found.
Of the substances which have been isolated directly from the brain, cere-
bron has been best studied, and its constitution established by Thierf elder.'
He obtained from it, by hydrolysis, cerebronic acid, sphingosine, and galac-
tose in the following proportions: cerebronic acid 48.13 per cent, sphin-
gosine 34.46 per cent, galactose 21.77 per cent.

Cerebron has the empirical formula C48H93NO9. Its hydrolysis takes
place in accordance with the equation:

C48H93NO9 + 2 H2O = C25H50O3 + CiyHssNOo 4- CfiHiaOs

Cerebron

Cerebronic acid Sphingosine Galactose

' F.Baumstark: Z. physiol. Chem. 9, 145 (1885).
» Liebreich: Annal. 134, 29 (1865).

' H. Thierfelder and Emil Worner: Z. physiol. Chem. 30, 542 (1900). H. Thierfelder:
ibid. 43, 21 (1904), and 44, 306 (1905).

614 LECTURE XXVI.

A number of other products containing nitrogen, and, for the most part,
phosphorus as well, have been isolated from the brain and described under
different names. We have no means of deciding at present anything
regarding the nature of these substances as to whether they are simple
substance or mixtures, and for that reason will not stop even to enumerate
them.'

Nervous tissue always contains cholesterol, fat, and albumin, and, in
fact, besides nucleoproteids and nucleoalbumins, globulin and albumin.
The framework of the tissue is formed by neurokeratin.

If we compare the amounts of the separate constituents present in 100
grams of organic dry substance, we note especially the high albumin con-
tent of the nervous tissue. Thus Chevalier - analyzed the sciatic nerve of
man (the central organs have a similar composition), and obtained the
following values:

Protein 36.8 per cent

Lecithin 33.57 per cent

Cholesterol 12.22 per cent

Cerebrin 1 1 . 30 per cent

Neurokeratin 3.07 per cent

Other organic substances 4 . per cent

It is a striking fact that gray brain matter contains much more
water than white brain matter. The former contains about 77 per
cent of water, while the latter has but 70 per cent. The proteins occur
chiefly in the gray matter, and amount to more than one-half of its dry
substance.

The metabolism of nervous tissue is almost entirely unknown to us.
Whereas in the case of muscles we are able to detect purely external
changes (shortening) during their activity, and at the same time can detect
the liberation of heat and consumption of glycogen, this is impossible in
the case of nervous tissue. In the ganglion cells alone have histological
pictures been described which apparently indicate changes. The nature
of these changes is, however, very obscure. We merely know that nervous

' As regards the chemical constituents of brain, see J. L. W. Thudichum: Die chem-
ische Konstitution des Gehirns des Menschen und der Tiere. Tubingen, 1901. Although
the investigations of Thudichum are in certain respects valuable as a basis for further
physiological-chemical investigation, still it must be said that none of the many com-
pounds described by him was proved to be a simple substance, nor was there given any
proof concerning the chemical combination of the substance. There is an almost
unexplored field for investigation here. The article by W. B. HaUiburton, Die Bio-
chemie der peripheren Nerven. Ergeb. Physiol. (Asher and Spiro) Jg. 4, p. 23
(1905), is a valuable summary of the work that has been done.

« Z. physiol. Chem. 10, 97 (1886).

RELATIONS OF THE ORGANS TO ONE ANOTHER. 615

tissue requires oxygen for the performance of its functions. This may be
shown very prettily by means of methylene blue. As we have seen, this
dyestuff is deprived of oxygen by the tissues, and is thereby transformed
into the colorless reduction product. If the colorless organ is subse-
quently exposed to the air for some time, gradually the blue color of
methylene blue reappears. In the case of narcotized animals in which
the brain has been made inactive, the brain substance remains blue, so
that evidently under these conditions the tissue doee not require oxygen.'
The abundance with which the nerve-centers are supplied with blood-
vessels is an indication of their high oxygen requirement, and in fact
anaemia in these places causes severe disturbances, eventually leading
to the loss of function.

It might be thought that some idea concerning the metabolism of nervous
tissue might be gained by seeking the end-products. As a matter of fact,
in the cerebro-spinal fluid, which in a sense may be regarded as the lymph
of the brain, choline, a decomposition product of lecithin, has been found.
This, however, does not give us much information. We do not know
whether choHne is to be regarded as a true metabolic end-product, or
whether it is not more probably produced by the destruction of nerve-
tissue, and is far from being concerned in true metabolism. Its detection
has usually been an indirect one and not quantitative. The fact that an
increased amount of choline appears in degenerative processes indicates
that its formation corresponds to a destruction of nervous tissue, so that
it is not to be considered as a normal metabolic product. We do not wish
to place any great weight upon this discovery of the presence of choline,
and would rather take the attitude that at present we know nothing what-
ever concerning the metabolism in nervous tissue. It is also hardly to be
expected that there will be much progress in this direction for a long time.
There is no foundation for research. Even our knowledge of the compo-
sition of nervous tissue is extremely faulty. We have only to remember
that, with few exceptions, we know but little concerning the metabolism
of those cells of the body which are much more readily accessible. We
recognize only the total results of metabolism, and are, as a rule, ignorant
as regards the part taken by the separate organs. We shall soon come
back to the fact that it is impossible to detect any effect of intense mental
effort in metabolism experiments.

We might perhaps expect that pathology would tell us something
concerning the metabolism in nervous tissue. We know that there exist the
so-called functional nervous diseases, i.e., diseases which, according to the
general assumption, are not caused by any anatomical change of the

' C. A. Herter and A. N. Richards: Am. J. Physiol. 12, 207 (1904). Cf. H. von
Bayer: Z. allg. Physiol. 2, 169 (1902). Frohlich: ibid. 3, 131 (1903). Frohlich and Tait:
ibid. 4, 105 (1904). K. H. Baas: Pfluger's Arch. 103, 276 (1904).

616 LECTURE XXVI.

nerve-tissue. We observe all sorts of different symptoms, one of the most
characteristic being the readiness with which the patient becomes fatigued.
One gets the impression that the nerve-centers have but a limited supply
of material at hand, or that the combustible material is consumed rapidly
without a sufficient regulation. This, however, is merely supposition
and rests upon no foundation. In the case of organic nervous diseases,
the symptoms of which are characteristic according to the nerves and
nerve-centers that are affected, we find degeneration. The nerve-cells
and their processes are destroyed. We may say that the experiment
has been tried, to trace some relation between the selection which certain
poisons, e.g. lead and "syphilis poison," show toward the various nerve-
paths and the activity of the metabolic processes in special regions.
Those nerve-paths and nerve-centers are assumed to show soonest the
action of the poison which are most affected because they have the most
work to do. As interesting as this hypothesis of Edinger ' may be, we
must state that there is absolutely no direct proof possible at present.
As long as the physiological course of metabolism remains so obscure, it
is very difficult indeed for us to get any clear idea from pathological
deviations. We can, it is true, imagine that cells which are in constant
activity and are constantly being used up and reconstructed, will feel the
effects of a given poison much more rapidly than those which are fairly
stable. This, however, does not npcessarily imply that there is an exhaus-
tion in the sense meant by Edinger, but rather it may be that there is an
effect upon the more active supply of new material and increased con-
sumption which is necessary for the exercise of the enlarged function.
We know that there are certain cells, e.g., of infusoria, algae, etc., which
have a particular power of attracting certain metals, even when the latter
are present in extremely slight amount. They take up these substances
even from very dilute solutions and store them up. This may very easily
cause the destruction of the cells, evidently in part, at least, on account of
the fact that these substances combine with the constituents of the cell
in such a way that they prevent the further exerci.se of function by the
cell-protoplasm, and to some extent destroy the cell. Similarly it is
conceivable that the interposition of the above-mentioned poisons between
the separate constituents of the protoplasm of nerve-centers, disturbs the
normal construction of the cells, and, therefore, its functions. There is
not necessarily any affinity existing between the nerve-cell as a whole,
and the poison in question, for it is perfectly possible that in the breaking
down and building up of the constituents of protoplasm, the products
resulting combine with the poison and consequently place a limit upon

' Eine neue Theorie uber die Ursachen einigen Nervenkrankheiten insbesondere der
Neuritis und Tabes, Leipsic, 1899, und Deut. med. Wochsch. Nos. 45, 49, 52 (1904) ;
Nos. 1 and 4 (1905).

RELATIONS OF THE ORGANS TO ONE ANOTHER. 617

the normal composition of the cell-protoplasm. We mention these ideas
merely to show that we are able to get along perfectly well without the
conception of exhaustion. On the other hand, we must remember that
the various nerve-cells are not necessarily all of uniform nature. It is
perfectly possible that the different groups of nerve-cells, which serve for
the exercise of definite functions, are differently constituted chemically
and possess a perfectly distinct metabolism, and that the key to the
cause of the different diseases of the system is to be sought in this fact.
The familiar diseases of the nervous system which are always, within quite
narrow limits, localized along definite paths are of quite particular interest
in many directions. Here the close connection between nervous and
muscular tissue becomes very evident.'

We have repeatedly spoken of the direct and indirect influence of the
nervous system upon all the organs of the body. It is quite impossible to
define this more closely, i.e., we cannot in any way explain the nature
of the conveyance of sensation. We merely know that the nerve-fibers
are merely outgrowths of the nerve-cells with which they form a unit.
The former are in contact with the organs usually by means of a character-
istic end-apparatus. It does not seem at all impossible that physiological
chemistry, together with physical chemistry, will eventually throw light
upon this process. It can hardly be doubted that definite changes,
whether in the end-apparatus or in the nerve-cells, give rise to definite
functional expressions of the nervous tissue.

Before we take up the relations of the musculature to the other organs,
we will consider briefly a reaction which is common to both muscle and
nervous tissue, namely the so-called heat-rigor.'^ If a muscle is gradually
heated, it loses at a definite temperature its power of being stimulated,
and contracts. The cause of this heat-rigor is the coagulation of the
albumin, and it may be shown that this does not take place all at once, but
in stages. Different albumins present in the muscle coagulate at different
temperatures. The nerves behave in exactly the same way. They also
show a heat-rigor. The lowest temperature at which one of the albumins
coagulates, corresponds to the time that the nerve ceases to be capable
of response to stimulation. Here, as in the case of muscles, there is a
contraction.

As is well known, the albumins in muscles coagulate after death. Rigor
mortis results, and this does not attack all the different muscles at one
time. Its appearance after death also occurs at different intervals of
time. The cause of death-rigor has been much studied. It is certain

' Cf. Robert Bing: Deut. Arch. klin. Med. 85, 199 (1905); and Deut. Z. Nervenheilk.
26, 163 (1904); and Ueber angeborene Muskeldefekte. Inaug. -Dissert. Basel (1902).

^ Brodie and Richardson: Philos. Trans. London, Series B, 191, 127 (1899). Vernon:
J. Physiol. 24, 239 (1899). O. von Furth: Z. physiol. Chem. 31, 338 (1900).

618 LECTURE XXVI.

that it results from the coagulation of the protein in muscle, and in fact
it is believed to be the protein known as myogen which takes part especially
in the process. This is supposed to pass over first into soluble myogen-
iibrin, which is subsequently transformed into the coagulated modification.
The other protein, myosin, also takes part in the formation of the clot.'
Although the assumption that death-rigor results from a coagulation of
the protein has been generally accepted, on the other hand the manner
in which the coagulation takes place has been variously explained. Con-
siderable attention has always been paid to the fact that an acid reaction
appears. This is apparently brought about by the formation of lactic
acid, and by the resulting transformation of a part of the diphosphate
contained in muscle to monophosphate. Now it has been observed that
acid, and especially lactic acid, accelerates the coagulation process. The
early appearance of death-rigor after previous active muscular contraction,
as, for example, in tetanus, has been attributed to the action of the lactic
acid, which is in such cases present in larger amount than usual. Further-
more, it has been assumed that a ferment assists in causing the coagulation.
The final relaxation, which takes place after an indefinite length of time,
is also not perfectly understood. Acids have been supposed to take part
in this process also, while, on the other hand, it has also been assumed
that autolytic processes come into play.

In certain directions we are well informed concerning the processes of
metabolism which take place in muscles during the exercise of their func-
tions. We have already discussed these points. We also know that the
liver is apparently in direct relation to the muscles, for, by means of its
glycogen store, it satisfies their nutritional requirements. It is not neces-
sary that the relation between the liver and muscles should be a direct
one. It may be brought about by means of the blood. This has, as we
know, a sugar content which is very constant within narrow limits. In
case the sugar in the blood is used up by the muscles, it receives a new
supply from the liver, the cells of which in such cases at once break down
glycogen into sugar. There must also, without doubt, be relations between
the general metabolism of the cells and that of the muscles. If, for example,
it is desired to fatten with albumin, this can be accomplished well only
when there is muscular effort at the time the abundance of albumin is fed.
This may be explained on the assumption that under such conditions
albumin is assimilated to a greater extent than usual, although it is also
possible that the other cells of the body are in some way stimulated to
take up more albumin.

This finishes all that we have to say here with regard to the relations of
the individual organs to one another. They are, to be sure, much more

' O. von Fiirth: Arch, exper. Path. Phar. 36, 231 (1895).

RELATIONS OF THE ORGANS TO ONE ANOTHER. 619

diversified than we have here represented, and are of great importance in
the study of the processes which taije place in the animal organism. It
is one of our most important tasks to follow these problems farther. It
is only when we shall have acquired as thorough a knowledge as possible
in this direction, that we shall be in a position to draw a clear picture of
the cell-metabolism and the functions of the organs. The dependence
of the organs upon one another is, as a rule, too little emphasized.

LECTURE XXVII.

GENERAL METABOLISM.

I.

We have so far considered for each foodstuff the way it is absorbed,
assimilated, and finally eliminated from theanimal organism, and attempted,
above all else, to follow the intimate processes of metabolism in the tissues
and especially in the cells. The study of these processes separately has
to-day become the particular field of the physiological chemist. We should
err greatly, however, if we were to regard the chemical decompositions in
the animal organism solely from the standpoint of the individual food-
stuff. We should obtain an entirely false impression of the general meta-
bolism, and should be unable to answer some of the most important
questions. We have up to this time studied metabolism, as it were, from a
more or less qualitative standpoint. There remains the quantitative side
to be considered; i.e., we must compare the total income and the total outgo.
We have already touched upon this problem in discussing the transforma-
tion of the different organic foodstuffs into one another, and in considering
their mutual replacement according to their calorific value.

It is particularly important for the study of metabolism that in the
economy of the animal organism the law of the conservation of matter
and of energy holds absolutely. This fact forms the basis of all experi-
ments in metabolism. We may determine, by studying as accurately
as possible the income and outgo, the part played by each individual
foodstuff in the general metabolism. Only by comparing the income
with the outgo are we able to form judgment as regards the condition of
the system. In this way alone is it possible to determine whether the
animal experimented upon increases its balance of nutrition, is in nutritive
equilibrium, or whether there is a deficit such that the organism is com-
pelled to draw upon its reserve stores or to consume its own tissue in order
to maintain the functions of its organs. The study of the body-weight
alone can never take the place of this important method of examination.
An increase or loss in weight may arise from a number of different causes.
Such deviations, for example, may be brought about merely by a retention
or an elimination of considerable water. The varied natures of the
problems concerning metabolism have led to different methods of investi-
gation. In some cases it is sufficient to follow the course of a single food-

620

GENERAL METABOLISM. 621

stuff, while at other times it is necessary, in order to draw a clear picture,
to measure the total intake and outgo. A simple problem, for ex-
ample, is to determine whether a definite substance acts as an albumin
sparer. Here, in most cases, it is sufficient to estimate the amount of
protein in the food without introducing any serious error, by merely de-
termining the amount of nitrogen contained therein. The amount of
nitrogen in the urine and in the faeces then shows clearly whether the
animal experimented upon is in nitrogen equilibrium or not. If the
animal is once found to be in such equilibrium, i.e. eliminates the same
quantity of nitrogen that it receives in the food, then bj^ feeding the given
food we can easily determine whether the elimination of nitrogen is in-
creased, diminished, or remains the same. The experiment in this case is
so simple, because we know that nitrogen is given up by the kidneys and
not by the lungs or skin. Such an experiment, however, is not perfectly
satisfactory. A number of questions always arise concerning such results.
We are by no means justified in assuming that the appearance of nitrogen
in the urine is a sign that there has been a total consumption of the albu-
min. We know that in all cases only a part of the carbon appears com-
bined with nitrogen in urine. The rest of the carbon chains from the
cleavage-products of proteins are broken down in a different manner.
The.se chains may remain in the organism long after all of the nitrogen
has been eliminated, and take part in metabolism in a way which is not
yet clear to us. At all events, in an exact investigation it would be neces-
sary to take into account also the elimination of the sulphur. But even
here we cannot be entirely satisfied. Only by combining the examination
of the urine and fseces with that of the remaining elimination products,
especially the gaseous ones, shall we obtain an exact insight into the
influence upon the total metabolism. In many cases even with such
experiments the results are not entirely satisfactory. We should know
oftentimes more accurately to what extent kinetic energy has been changed
into potential energy, in order to judge correctly the physiological
nutritional value of the individual foodstuff.

Before considering the details of an experiment in metabolism, we
must bring forward the fact that an exact insight into the questions
concerning metabolism can only be expected when influences which have
no bearing upon the problem at hand are excluded as completely as
possible. Comparative experiments in which the basal conditions are as
nearly alike as possible, should be carried out as a rule upon the
same animal. Individual peculiarities which sharply influence metabolism
should never be disregarded. One of the most important requirements to
be satisfied in a metabolism experiment is that the test should be carried
out for a considerable length of time. It is not possible to draw any
exact conclusions from observations made only during a period of twenty-

622 LECTURE XXVII.

four hours or less. The metaboHsm of the food eaten the previous day
will affect the results; and, moreover, the metabolism of the food taken
during the day of the experiment will not be complete during such a
short time. Atwater ' showed how much more valuable the results were
when the experiment was continued for some considerable time. A
great many contradictions and differences in the researches concerning
metabolism, which are to be found in the literature, may be traced to the
fact that this requirement has not been satisfied.^

As a foundation for a metabolic balance, an accurate knowledge of the
composition of the income and of the outgo is essential. As regards the
income, the foods are to be considered in two directions. We can, on
the one hand, evaluate them according to their chemical composition,
and, on the other, according to the energy which they contain. We obtain
the former values by means of chemical analysis. We have the organic
and inorganic constituents to estimate. In the former the carbon, hydrogen,
and nitrogen are determined. By multiplying the nitrogen found by 6.25,
the amount of albumin is obtained. Of course this method of estimat-
ing the amount of albumin is not an exact one. It is perfectly possible that
the food may contain nitrogen in some other form than as albumin. In
general, however, the amount of such nitrogenous matter is inconsiderable.
The fat content is obtained by extracting the food with ether, in which
connection it is to be remembered that a part of the fat will go into solu-
tion only after the product examined has been " opened up " ^ by digestion
with pepsin-hydrochloric acid, or with two per cent hydrochloric acid.
Then, when the ash is known, the amount of carbohydrate may be
determined by difference. By drying an aliquot part of the weighed
mixture, and weighing the dry residue, the amount of water in the food is
determined. Oxygen is naturally also to be considered among the sub-
stances taken into the system. Unfortunately, up to the present it has
not been found possible to measure accurately the amount of this gas
that is utilized. This thwarts the exact answering of many questions
in the field of metabolism. The potential energy of the food may be
ascertained by the heat of combustion. In exact experiments we must
naturally also take into consideration the temperature of the food and
drink as it is taken into the system.

' Ergeb. Physiol. (Asher and Spiro) Jg. 3, 497 (1904).

' Studies of metaboli.sm under pathological conditions are important. Clinical
investigation and experimental pathology are closely connected with the progress of
the knowledge of metabolism under normal conditions. By such means many new
questions have arisen, and certain disturbances have given us insight into this or that
process. It would be beyond the scope of these lectures to attempt to mention the
numerous discoveries which have been made in this way. We can give only the outline
here and the more important results. The physiology of metabolism has become such
an important branch of science that it can be studied only upon a broad basis.

» Cf. Lecture XIV, p. 325.

GENERAL METABOLISM. 623

The income is to be contrasted with the outgo. There are three prin-
cipal waj's in which the products of metabolism leave the organism, —
through the kidneys, the intestine, and the lungs. The faeces contain
not only products of metabolism, but also the unabsorbed food. The
determination of the nature of the fieces is highly important. It gives us
an idea how completely the nourishment has been utilized. In the urine,
besides the inorganic salts, we have to consider the nitrogen, sulphur,
phosphorus, carbon and hydrogen content. The first three elements give
us information concerning the decomposition of the protein. Usually the
nitrogen is alone determined and the amount of consumed protein is
obtained by multiplying the nitrogen value by 6.25. In many cases the
decomposition of the protein is also traced qualitatively, and it is deter- '
mined how much nitrogen is present as urea, and how much in the form
of other compounds. In order to get an idea of the energy economy, the
heat of combustion of the entire excreta is determined, further the heat
given off by the body, and the amount of heat equivalent to the muscular
work performed.

The gas metabolism is studied with the help of apparatus of special
construction,' by means of which the amounts of carbon dioxide and
water vapor eliminated are determined. From the amount of carbon
dioxide the equivalent weight of carbon may be computed. This, together
with the amount of carbon contained in all the remaining excreta, gives us,
first of all, an idea as to the utilization of the carbon in the food by the
organism. The question then arises how can we tell from the total amount
of carbon the amount that has resulted from the separate foodstuffs,
albumin, carbohydrate, and fat. To estimate this we start with the total
amount of nitrogen eliminated, and from that compute the amount of
albumin decomposed. Since the average carbon content of protein is
known, it is easy to compute how much of the carbon came from proteins.
This is naturally on the assumption that the combustion of the remaining
protein molecule takes place simultaneously with the elimination of the
nitrogen. As a rule, the relation of nitrogen (16 per cent) to carbon (53
per cent) in protein is as 1 : 3.3. If, then, we multiply the nitrogen value
by 3.3 we obtain the amount of carbon which was obtained from protein;
and by deducting the product from the total amount of carbon in the
egesta, the amount obtained from nitrogen-free food is given. If there
is no remainder, then only protein was consumed. By comparing the total
amount of carbon and the remainder after deducting the carbon from

' Descriptions of such apparatus may be found as follows: Regnault and Reiset:
Annal. 73, 92, 129, 257 (1850), and Ann. chiin. et phys. (3) 26 (1849). Hoppe-Seyler:
Z. physiol. Chem. 19, 574 (1894). Pettenkofer: Annal. II, Suppl.-Band, p. 1 (1862).
Voit: Z. Biol. 11, 541 (1875). Sond^n and Tigerstedt: Skand. Arch. Physiol. 6 (1895);
and Atwater: Ergeb. Physiol. (Asher and Spiro) Jg. 3, 498 (1904).

624 LECTURE XXVII.

protein in the egesta with that in the ingesta, we can tell whether all of
the carbon has been eliminated or whether more or less. In the two latter
cases we can tell whether the organism has consumed its own protein or
fat, or whether it has added to its supply of albumin and of nitrogen-free
substances.

We have disregarded the losses which the body sustains by its secretions,
and the losses of epidermis from the skin, intestinal canal, and other mucous
membranes. These losses are very hard to estimate. To some extent they
come into consideration with the eliminations from the alimentary canal
in the analysis of the fseces. They may be disregarded, or not be estimated
by themselves, because they are so small in amount that the error intro-
duced does not assert itself in the nutrition balance. Particular attention
should be paid to the nitrogen content of the fseces, which arises largely
from intestinal depositions. The fseces of man contain 0.5 to 1.4 grams of
nitrogen, even when the food contains but little nitrogen or none. Among
the nitrogen-free products, the fseces contain principally fat. In starvation
man eliminates 0.6 to 1.4 grams of fat per day. With nourishment free
from fat, 3 to 7 grams are eliminated daily.

For the determination of the heat given off by the organism, a special
apparatus is also necessary. A calorimeter which permits the simultaneous
determination of the respiratory exchange and the amount of heat liberated
is Atwater's respiration calorimeter.^ This apparatus, in which the person,
or animal, experimented upon may remain for a week, consists of a chamber
which is large enough to be comfortable. This space is supplied with a
ventilating arrangement, .so that the volume of the air can be accurately
measured. It is so regulated that the air enters and leaves at exactly the
same temperature. Now antl then samples of air are taken as it enters and
as it leaves the chamber, whereby the amount of carbon dioxide and water
given off by the lungs and by the skin is determined. Suitable arrange-
ments are also provided for the introduction of food and drink, and for
the removal of the solid and liquid excretory products. The amount of
heat given up by the organism, together with the heat equivalent
of the external muscular work, is measured at the same time. The heat
given off is carried away by a stream of cold water, which flows in tubes
around the chamber. By carefully regulating the temperature of the water
and the velocity at which it flows through the tubes, it is possible to carry
away the heat as fast as it is produced, thereby keeping the temperature
of the chamber constant. By determining the amount of water that has
flowed through the tube, and the change in temperature, it is possible to
estimate the amount of heat given off by the body.

By determining the amount of water vapor in the air as it enters and as it
leaves the chamber, it is possible to find out how much water is given off by

' Loc. cit. p. 499.

GENERAL METABOLISM.

625

the body. The amount of heat energy required for its evaporation must be
added to the amount of heat energy carried away by the water current.
Atwater measured the heat equivalent of the external work performed in
a very interesting manner. He provided a bicycle which was connected
with a dynamo. The electric current produced was led through an incan-
descent lamp, and there transformed into heat energy, which can be directly
measured as such. From the duration of the experiment, and the amount of
electric current produced, the amount of work performed can be estimated.

According to this method of investigation, the kinetic energy of the
body is computed from three factors. First, there is the total amount of
heat carried away as heat, including that carried away by the air current ;
second, the latent heat of the water vapor which is carried away from the
body; and third, there is the heat equivalent of the external work. The
first amount of heat has several sources. It comprises the heat given off
by radiation and conduction from the skin, that obtained from the excreta
on their being cooled to the room temperature, and further there is
the cooling of the expired air (carbon dioxide and water vapor) to the
room temperature.

By means of such an apparatus not only the influence of different kinds
of nourishment upon metabolism can be determined exactly, but also
their relations to the external work. As we have already seen,' it is by
the help of such experiments that we have been able to prove that the
fats, in order to be utilized for muscular work, need not necessarily be
transformed into carbohydrate, but that their calorific value may be
applied directly.

Name, Nature of tbe
Experiment.

Laboratory

Number

Duration
in Days.

Transformed Energy.

Excess
Energy in
Period of
Work Over
Period of
Rest.

Heat
Equiva-
lent of the
External
Work.

Utiliza-
tion in
Per cent.

E.O.
Rest experiment
Work experiment

J. F. S.
Rest experiment
Work experiment

J.C. W.
Rest experiment
Work experiment
Minimum work
Maximum work
Average

4

2

14

42
12

12

18

4

16

8

46

2279
3892

2119
3559

5056
5332
6143

1613
1440

2699
2975
2786

529
601
646

13.3
16.2

19.8
20.2
19.6

See Lecture XV, p. 338 et seq.

626 LECTURE XXVII.

Atwater succeeded in solving a number of important problems concern-
ing metabolism with the help of his apparatus. If we consider metabo-
lism simply from the standpoint of energetics, i.e., consider the organism
simply as a machine, then one of the first questions to interest us is as
regards the utilization of the food for the performance of external work.
We must remember that an ordinary steam engine on an average converts
but 15 per cent of the energy contained in the fuel into work. The rest
is set free as heat.

The table on page 625 shows the relation of the external muscular work
to the total amount of transformed energy, from which the efficiency of
the human body as a machine may be computed.'

The work performed in these experiments was measured by the bicycle
arrangement described above. In the table its heat equivalent is taken
into consideration. The objection might be raised that it is hardly possible
to distinguish sharply between a period of rest and one of work, for in the
latter case there is merely additional work over that required by the
organism for the exercise of the remaining physiological functions. It is
difficult, and in fact entirely impossible, to make sure that the mental and
nervous expenditure of energy will be exactly the same in two experi-
mental periods. A priori it is conceivable that there is a considerable
transformation of energy in these two kinds of work. To meet this objec-
tion Atwater - compared the results obtained where the person experi-
mented upon was resting mentally and physically as completely as possible,
with results obtained in a period where the person was engaged in severe
mental effort, and found that there was no appreciable increase in the
transformations of matter or of energy in the latter case. This does not
necessarily mean that mental activity does not correspond to a considerable
consumption of energy. It is entirely impossible to stop completely at
will all mental effort. The mental work continues, whether the person
experimented upon, as in Atwater's experiments was the case, is busied
with the results of experiments, a German treatise, or the study of physics;
there is no apparent difference from the results obtained in metabolism
when the brain is as much at rest as is possible to make it voluntarily.
As far as the above experiments are concerned, therefore, it makes no
difference to what extent brain and nervous activity affect the total
consumption of energy; for, as Atwater has shown, the consumption of
energy from these causes remains the same, whether mental work is per-
formed intentionally or unintentionally. A glance at the above table
shows that all human organisms do not work with the same degree of effi-
ciency. That of E. O. was less efficient than that of J. S. F. and J. C. W.
At all events, however, as a machine the human organism is more efficient

' Atwater: loc. cit. p. 608.

' Atwatei-: U. S. Dept. Agr. Office of E.xper. Stations. Bull. 44.

GENERAL METABOLISM. 627

than an ordinary steam engine. We must admit, on the other hand,
that the way this was proved by Atwater's experiments is not altogether
beyond reproach. It is not an exact determination. We do not know
just how much effect the increased muscular work had upon the functions
of the other organs. At the same time it is probable that the values
obtained are not far from the truth.

While the advance in technique and in methods results in more precise
investigations, stUl, on the other hand, we are often compelled to resort
to indirect methods with all their sources of error which we have so often
mentioned. This is true in many cases when we attempt to determine
the participation of nitrogen-free foodstuffs in metabolism. We can, it
is true, get some idea of this bj^ comparing the volumes of inspired and
expired air. In normal breathing the volume of the expired air is always
greater than that of inspired air. This is due to the fact that the outer
air is warmed to the body temperature after it is inspired, and at the same
time it is almost completely saturated with water vapor. In order to
obtain actually comparable figures it is necessary to measure the two
volumes of air under precisely the same conditions. Both must be brought
to the same temperature and pressure; and, furthermore, they must be
dried. When this is done, it will be found that the volume of the expired
air is almost invariably smaller than that inspired. This is due to the
fact that in the combustion of the foodstuffs, the carbohydrates alone
yield a volume of carbon dioxide equal to that of the oxygen consumed,
while in the combustion of protein and fat this is not the case. In the case
of the latter, a part of the inspired oxygen is utilized for the formation of
water, sulphuric acid, and other substances. The amount of oxygen con-
sumed in this way does not appear in the volume of the expired air when
measured as above. In the combustion of carbon, one volume of carbon
dioxide gas is formed from one volume of oxygen. In this case the ratio

—T~- = 1. This relation of expired carbon dioxide to inspired oxygen is

called the respiratory quotient. In the combustion of carbohydrates
this is 1. A diet in which protein predominates causes the quotient to
fall to about 0 . 80, while if fat is chiefly concerned in the metabolism, the
value of the quotient falls to 0.70. According to the value of this re-
spiratory quotient, therefore, we can draw some conclusions regarding the
nature of the food upon which the subject experimented upon is working.
If, for example, a dog which has a good supply of glycogen stored up is made
to fast, then the high respiratory quotient, which is approximately 1,
shows that at the given moment the dog is maintaining its economy
chiefly by drawing upon its carbohydrate stores. When the value of the
quotient begins to fall, it shows that fat is being consumed, and finally it
will be compelled to utilize its own protein. Naturally, it is not, in general.

628 LECTURE XXVII.

advisable to depend upon this respiratory quotient alone, but we should
also take into consideration the other eliminations, especially that of
nitrogen.

Now that we have roughly sketched the outlines of the methods employed
for studying metabolism, we will briefly discuss, before taking up the
important facts that have been ascertained concerning metabolism under
definite conditions, the influence of the conditions created by the subject
experimented upon itself. First of all, there is the question of size. It
is perfectly clear that the total metabolism will be more extensive in pro-
portion to the size of the organ in function. Eventually the consumption
of material is to be traced to the work of the individual cells, and the more
cells there are the greater will be their total requirement. Thus a small
animal will require absolutely less nourishment than a larger one. Of
course individual peculiarities play a part which must not be left out of
consideration. If, on the other hand, instead of paying attention to the
absolute amount of material consumed, we consider the energy trans-
formed per kilogram of body-weight, provided we are working under
otherwise parallel conditions, we shall find that the metabolism of the
smaller animal is greater than that of the larger one. In order to obtain
values which shall be actually comparable, the separate experiments upon
metabolism must be carried out with the animal at rest, and also fasting.
In this way the so-called fasting value is obtained.' The reason that
a small animal decomposes more substance in proportion to its own
weight lies in the following: — The smaller an animal is, the larger the
surface of its body in comparison to the volume and weight of the body.
It may be assumed that about four-fifths of the total heat given off by
the body is through the skin. The amount of heat lost by the skin is very
nearly proportional to the amount of surface covered by it, so that the
smaller animal with its relatively larger surface loses more heat than the
larger animal. Consequently the smaller animal requires a greater supply
of heat energy than the larger one, as otherwise its body temperature,
which is regulated by two factors, the amount of heat generated and that
given ofT, will not be maintained at the proper height.

The tables on the following page show how the amount of oxygen
consumed depends upon the size of the body,^ and give also a comparison
of the metabolism of energy in animals of various sizes with their relative
surface development.'

The influence of the greater surface becomes apparent when we compare
the metabolism of younger and older individuals of the same species.

' Cf. Max Rubner: Z. Biol. 19, 535 (1883). Slowtzoff: Pfluger's Arch. 96, 158 (1903).
Karl Oppenheimer: Z. Biol. 42, (1901).
' Max Rubner: Z. Biol. 19, 536 (1883).
' Max Rubner: ibid. p. 549.

GENERAL METABOLISM.

629

This, however, of itself does not by any means explain the considerably
more active metabolism on the part of the younger individual. This is
evident when we compare not the metabolism of the whole external sur-
face, but rather that per unit of surface. On comparing, for example,
the metabolism per square meter in fully developed dogs of different sizes,
we will find that the value is the same for all dogs within certain narrow
limits. This is, however, not the case if we compare the metabolism of
young dogs with that of older ones. In the case of the former the metabo-
lism is greater per unit of surface than it is in the older dogs.

I.

CONSUMPTION OF OXYGEN, ARRANGED IN THE ORDER OF THE
ANIMAL'S WEIGHT.

Grams Oxy-

Grams Oxy-

Species.

Weight in

gen Absorbed

Species.

Weight in

gen Absorbed

Kilograms.

per Kilogram

Kilograms.

per Kilogram

in 1 Hour.

in 1 Hour.

Male calf . .

115

0.481

Rabbit . . .

3.43

0.735

Male calf . .

115

0.428

Mountain rat

1.55

1.198

Sheep ....

70

0.464

Hen

1.51

0.846

Sheep ....

66

0.490

Drake. . . .

1.22

1.382

Sheep ....

65

0.400

Cross-bill . .

0.028

10.974

Hen-turkey .

6.2

0.702

Green-finch .

0.025

13.000

Dog

5.59

0.902

Green-finch .

0.025

9.742

Goose ....

4.60

0.677

Sparrow. . .

0.022

9.595

Rabbit . . .

3.58

0.763

II.

COMPARISON OF THE EXCHANGE OF ENERGY IN ANIMALS (DOGS)
OF DIFFERENT SIZES WITH THEIR RELATIVE SURFACE DEVEL-
OPMENT.

Number.

Weight in
Kg.

Surface in
Cm=.

Surface in Cm-,
per 1 Kg. Weight.

Calories per 1 Kg.

in 24 Hours at

15° C.

Calories per 1
Cm-. Surface.

I.

31.20

10.750

344

35.68

1036

II.

24.00

8.805

366

40.91

1112

III.

19.80

7.500

379

45.87

1207

IV.

18.20

7.622

421

46.20

1097

V.

9.61

5.286

550

65.16

1183

VI.

6.50

3.724

573

66.07

1153

VII.

3.19

2.423

726

88.07

1212

In the case of sucklings the metabolism per kilogram of body-weight
i§ likewise much greater than it is with fully developed dogs; but, on the

630 LECTURE XXVII.

other hand, that per square meter of external surface is smaller than in the
case of the older dogs. This is not remarkable. The suckling performs
as a rule but little external work. It sleeps the greater part of the time,
so that the consumption of material is not so extensive as during latter
periods.

In old age, the metabolism is greatly diminished, even when we compare
the amount per square meter of external surface with the values obtained
in the same way at middle age. In human beings between the ages of 22
and 56 years the amount of carbon dioxide eliminated in an hour per square
meter of body surface is about 11.2 grams, while in old age (70 to 77 years)
the value is only 9.2 grams in the case of males; with females the elimina-
tion between the ages of 17 and 40 years is about 11.75 grams, while at
71 to 86 years it is only 9.79 grams.'

The fact that the extent of metabolism in different periods of life is
of different intensity need not surprise us. In considering metabolism
as a whole, we must not forget that it is the sum of the metabolism
of innumerable units, the cells. In the developing tissue of youth
the transformations are unquestionably much more extensive than in
the adult organism. We know from a great many observations that the
organism soon accustoms itself to certain functions, and usually expends
more energy the first time that a demand is made, whereas, later on,
it accomplishes the same result much more economically and with the
expenditure of far less effort. It is indeed conceivable that the cells of
the adult organism learn to work more and more economically. At pres-
ent we are not in a position to study the metabolism of the individual cells,
i.e., that of the protoplasm. We obtain the impression, instinctively rather
than as a result of scientific investigation, that the cells of the individual
are not all equally efficient. This hypothesis gains form when we recall
the numerous cases of pathology whose etiology is paraphrased with the
conception of disposition. There is no doubt that the various diseases
of metabolism, such as diabetes, gout, rachitis, etc., are eventually to
be traced to a disturbance in the metabolism of individual cells. This
may affect a larger or smaller cell-complex, and the whole cell-work of the
individual may be affected apparently without our being able to explain
precisely the way in which the metabolism of such a weakened or weak
individual is changed. As long as the cell itself belongs in the domain
of the unknown, we cannot expect to be able to gain precise information
concerning the physiology and pathology of cells. While we must empha-
size the fact that at present the designation of a pathological derange-
ment of a cell-function, or of the metabolism as a whole of individual
cells, only represents a conception in accordance with our present knowl-

Magnus-Levy and E. Falk: Arch. Anat. Physiol. 1899. Suppl. 314.

GENERAL METABOLISM. 631

edge, still, on the other hand, the importance of cell-life, of the metabolism
of the cells and their functions, must not be overlooked in considering
the metabolism of the body as a whole.

Interesting glimpses into the course of general metabolism have been
obtained by studying the results under definite conditions. The metabo-
lism taking place after the complete withdrawal of the food has been
studied especially. Under this condition the animal lives at first upon
its own stored-up material, and finally attacks its own tissues. The
duration and the whole course of the metabolism during starvation depends
largely upon the condition of the body at the beginning of the experiment.
As soon as a certain definite fraction of the body-substance has been used
up, death takes place. Naturally the activity of the metabolism also
affects the duration of starvation period. According to the principles
enunciated above, therefore, we should expect, a priori, that young in-
dividuals would suffer from the withdrawal of nourishment much more
quickly than adults. Similarly in the case of animals which in general
have a lower metabolism, as with cold-blooded animals, they will sur-
vive starvation longer than will the warm-blooded animals. Dogs can
live without food for six weeks. Birds live on an average from 5 to
20 days, while fish and reptiles may survive for from 6 months to a year.
Even in the case of human beings, long periods of fasting have been
observed.'

The first marked change that results from the withdrawal of all nour-
ishment is the loss in weight of the body, which within a relatively short
time is followed by a loss in muscular power. The subject sleeps much,
and towards the end of the period of starvation is in a somnolent con-
dition. The whole metabolism of the animal diminishes simultaneously
with the loss in weight. If, however, the amount of material transformed
is compared to a kilogram of body-weight, it will be found that the metabo-
lism is only slightly changed from that of a well-nourished animal. In
a short time the fasting animal adjusts itself to a minimum metabolism,
which remains constant for quite a while. First of all the animal makes
use of its stores of carbohydrates and fat. The former are soon exhausted.
From the beginning of the starvation period, albumin is continuously being
decomposed. The amount of albumin which the fasting organism must
decompose in order to accomplish the necessary metabolism, depends
chiefly upon the amount of nitrogen-free substances which are present.
If the animal is able to consume a considerable amount of the latter, then

' L. Luciani : Das Hungem, Hamburg-Leipzig, 1890. J. E. Johannson, E. Lundgren,
Klas Sond^n. and Robert Tigerstedt: Skand. Arch. Physiol. 7, 29 (1896). C. Lehman,
F. Miiller, L Munk, H. Senator, and N. Zuntz: Virchow's Arch. 131, Suppl. 1 (1893).
R. Tigerstedt: Nordisk, Medic. Arch. No. 37 (1897). C. Voit: Z. Biol. 41, 113 (1901).
Siegfried Weber: Ergeb. Physiol. (Asher and Spiro) Jg. 1, Abt. 1, p. 702 (1902).

63:;

LECTURE XXVII.

the albumin in the organism is protected to a certain extent from con-
sumption. On the very first day of fasting, there is in the majority of
cases a noticealjly liigli elimination of nitrogen, and especially when the
food has been rich in proteins. The rise in the amount of nitrogen elimi-
nated, which in individual cases, as with rabbits, for example, reaches a
maximum on the third to the fifth day from the beginning of the fasting,
is taken as a guide for determining the time when the carbohydrate stores
have been exhausted; i.e., when the albumin-sparing factor has been
eliminated.' Moreover, this increase in the decomposition of albumin is
not a constant phenomenon, and evidently depends upon the species. In
the case of rabbits, especially, it is hard to determine the exact day when
the starvation period begins. These animals have an extremely volumi-
nous intestine, and particularly in the cjecum there is always a mass of only
partly utilized material upon which the animal may subsist for some time.
In the case of dogs, in general there is a uniform, slowly diminishing elimi-
nation of nitrogen." Voit has shown the influence of a preliminary diet,
rich in protein, upon the elimination of nitrogen during the first day of
fasting, by the following three experiments.' He determined the amount
of nitrogen eliminated daily. Before beginning the experiment the first
dog was fed daily with 2500 grams of meat, the second dog received 1500
grams of meat, while the third dog was fed with a mixed diet, poor in
proteins.

First day of fasting .
Second day of fasting
Third day of fasting
Fourth day of fasting
Fifth day of fasting .
Sixth day of fasting .
Seventh day of fasting
Eighth day of fasting

Nitrogen Eliminated in Grams per 24 Hours.

Experiment I.

Experiment II.

Experiment III.

60.1

26.5

13.8

24.9

18.6

11.5

19.1

15.7

10.2

17.3

14.9

12.2

12.2

14. S

12.1

13.3

12.8

12.6

12.5

12.9

11.3

10.1

12.1

10.7

A summary of the nitrogen elimination in a five-day fasting experiment
with men, is given by the following values published by Tigerstedt : *

' R. May: Z. Biol. 30, 1 (1S94).

' M. Kumagawa and R. Miura: Arch. Anat. Physiol. 1898, 4.31.
' Voit: Hermann's Handbuch, 6, 1, p. 89 (1881).

' Robert Tigerstedt: Lehrbuch der Physiologie des Menschen. 3 Aufl. Bd. 1, p. Ill
(Leipsic, 1905).

GENERAL METABOLISM.

633

Decomposed in Grams.

Body
Weight in
Kilograms

Nitrogen.

Fat.

Carbohy-
drate.

Alco-
hol.

Transfor-
mation in
Calories.

67.8

23.41

87

267

28

2705

67.0

12.17

206

2220

65.7

12.85

192

2102

64.9

13.61

181

2024

64.0

13.69

178

1992

63.1

11.47

181

1970

64.0

25.44

64

250

22

2437

65.6

18.07

72

• 248

37

2410

Total
Transfor-
mation
per Kilo-
gram of
Body
Weight in
Calories.

Last day of eating
First clay of fasting
Second day of fasting
Third day of fasting
Fourth day of fasting
Fifth day of fasting
First day of eating
Second day of eating

39.9
33.2
32.0
31.2
31.1
31.2
38.1
36.8

From these value.s it i.s apparent that the starving man quiclcly adjusts
himself to a definite minimum consumption.

In the further duration of the fasting period the organism lives exclu-
sively at the cost of its protein and its stores of fat. The carbohydrates
are quickly consumed, and in the later periods come scarcely into
consideration at all. The starving organism is very economical with its
protein. Of the total calories 84 to 90 per cent come from the fat, and
only from 10 to 16 per cent from protein. This holds naturally only for fat
animals. After a time the stores of fat are exhausted, and the organism is
then compelled to obtain the requisite amount of energy (calories) at the
expense of its own protein. At this time there is a rapid increase in the
elimination of nitrogen through the urine. This was observed by Voit/
and has subsequently been much discussed. As a matter of fact, the
animals ex|}erimented upon were found to be not entirely free from fat at
the time when, just before death, there was an increased elimination of nitro-
gen in the urine. Often quite a considerable amount of fat has been
found to be pre.sent at such a time. On account of this fact the conclusion
has been drawn that the increased elimination of nitrogen is not
altogether due to the fact that the store of fat has been entirely con-
sumed, but that there must be other causes. F. N. Schulz ^ assumes
that the increased decomposition of protein just before death by starvation
is to be attributed to the sudden destruction of numerous cells in the body.
It is indeed conceivable that the cells, whose ability is taxed to the
utmost during starvation, in order to provide the necessary material for
the general metabolism, at last disintegrate. They are constantly giving

■ E. Voit: Z. Biol. 41, 113, 502, 550 (1901).

' F. N. Schulz: ibid. 41, 368 (1901), and Pfliiger's .\rch. 76, 379 (1899).

634 LECTURE XXVII.

up material but receive nothing from without. The increase in the elim-
ination of nitrogen shortly before death may be prevented by feeding
cane-sugar. Thus Kaufmann ' fed starving rabbits with 25 to 30 grams
of sugar daily, and when they died, at the end of 18 or 19 days, it was
without the appearance of the increased nitrogen elimination. This ex-
periment does not permit us to decide satisfactorily the question as to
the cause of the increased decomposition of albumin which has been so
often observed just before the end of the fasting period. It is possible
that, when the cane-sugar is fed to the starved animals, the destruction
of the cells is prevented. On the other hand naturally the carbohydrate
acts as a protein-sparer. Perhaps the increased elimination of nitrogen
is prevented for this reason. Furthermore, it must not be left out of
consideration that, as we have already frequently mentioned,^ there
is no question but that fat acts as a solvent for many substances, and
plays an important part in this direction, besides its function of acting
as reserve material. Especially in the extensive transportation of
material from one cell to another, it is possible that the fat content
of the tissues may play an important part. If the amount of fat
present has been reduced to a minimum, the e.xchange of material
will necessarily suffer, and thereby the entire metabolism. We must
also not forget that evidently the fat, as it lies deposited in the fat-
cells, cannot be consumed as such by the body. It must first be removed
from the cell. It is very proliable that a cleavage into fatty acid and
glycerol takes place. This eventually affects cell activity, for only the cell
can yield the required ferment. It is perfectly clear that when the cells
are working with a limited supply of material, the formation of the
ferment will finally be influenced. It might be thought, a priori, that the
oxidation power of the starved organism would likewise become consid-
erably lessened. This is, however, not the case, or at least not of the
oxidation processes as a whole. M. Nencki and N. Sieber^ injected sub-
cutaneously one gram of benzene into a rabbit weighing 2.517 kilograms.
They subsequently found 0.307 gram of phenol in the urine. The animal
was then starved for three days, at the end of which time it weighed 2.425
kilograms. Once more a gram of benzene was injected, and this caused
the ehmination of 0.334 gram of phenol. Analogous results were obtained
in e.xperiments with dogs. On the other hand, the observation that in
starvation the ratio of the neutral sulphur to the oxidized sulphur increases
in value indicates a lessened oxidation power of the starving organism.
We should not yet attempt to draw definite conclusions from the experi-
ments at hand. It is perfectly possible that the power of oxidation is

' M. Kaufmann: Z. Biol. 41, 75 (1901).

^ Cf. Lecture VI, p. 112.

' Pfluger's Arch. 31, 319 (188.3).

GENERAL METABOLISM.

635

lessened only in certain directions. On the other hand, in discussing
animal oxidations we called attention to the importance of the preliminary
cleavage for a complete combustion.'

If food is no longer withheld from the animal, it recovers rapidly.
It replaces first of all what it has lost of its own body-substance, and seeks
to regain its former condition.

Now it is very interesting to find that during starvation the animal
organism attacks the different parts of its own body-material to quite
different extents. It might have been expected, a priori, that those organs
would suffer most upon which the greatest demands are placed. On the
contrar}', there is a constant transference of material to those organs which
are most useful and are consequently most indispensable to life. This w'as
shown very clearly in considering the life and development of the salmon,^
and led to the question whether we must not assume that the starving
organism is obliged to effect extensive syntheses from the building-stones
of the less important cells. We can easily believe that the heart, whose
function is so essential for the maintenance of life, retains its material
unchanged and carries out its work at the expense of other tissue. On
the other hand, it is also possible that the muscle-cells of the heart are
constantly being broken down and rebuilt, ki present we have no means
of estimating the life of a cell, and cannot decide how long it can retain its
corporate existence, or whether it is constantly assimilating and giving up
material. If the latter is the case, then the tissues of the starving organism
must be the scene of remarkable transformations.

Voit ^ gives the following values for the loss of body-weight during starva-
tion in the case of doves and cats.

Per cent of Original

Per cent of Original

Weight.

Weight.

Doves.

Cats.

Doves.

Cats.

Fat

93

97

Testes

40

Spleen

71

67

Skin

33

21

Pancreas

64

17

Kidneys

32

26

Liver

52

54

Lungs

22

18

Heart

45

3

Bones

17

14

Intestine

42

18

Nervous system . .

2

3

Muscles

42

31

' Cf. Lecture XIX, p. 451 et seq.

' Cf. Lecture XVI, p. 351.

' Handbuch der Physiologic des GesamtstofFwechsels und der Fortpflanzung. Part I.
Physiologie des allgemeinen Stoffwechsels und der Emahrung by C. von Voit, Vol. 6,
p. 96, 97Leipsic (1881).

636 LECTURE XXVII.

It is evident from this summary that the organs which are in constant
activity, as the heart, lungs, kidneys, and nervous system, suffer much less
loss of weight than the other organs. Voit also tested the influence of the
activity of an organ upon keeping its composition constant by feeding
doves with food which was deficient in lime, but contained a sufficient
amount of other materials. A post-mortem examination showed that
those bones which were in constant use had suffered less from the lack of
lime than relatively inactive bones, such as the breast-bone and the bill.
The last two bones had become perfectly porous. Evidently the composi-
tion of the bones which were used most was maintained at the expense of
the others. In this direction the observation of E. Pfliiger on dogs is
worth mentioning. He found, as has already been mentioned,* that when
the pancreas was extirpated the fiver of these animals tended to gain
rather than lose in weight. Evidently the liver is an important place for
transformations, such as fat into sugar, etc.^

The loss in weight affects all the different substances contained in the
organs. The starving animal constantly loses water even when it is given
none to drink. Water is formed by the combustion of fat and albumin.
Mineral substances are likewise being eliminated constantly. The organs
maintain their functions as long as possible even in those cases where, as far
as we know, the organ is dispensable. Thus while the secretion of bile
diminishes, to be sure, during starvation, still, on the other hand, it con-
tinues to form for quite a length of time. Likewise the secretion of milk
continues for a time. On the other hand, the gastric secretion soon ceases,
as was shown by the examination of the contents of the stomach of the
fasting professional, Succi.

A very important result of these starvation experiments is that the
animal organism constantly eliminates nitrogen under all conditions. To
be sure, the amount of decomposed albumin may become quite small, but
it never ceases altogether. Albumin assumes, as we have already shown
in discussing the Law of Isodynamics,' a pecufiar position among our
organic foodstuffs. It cannot be entirely replaced by any other material.
It is possible to nourish a dog upon albumin alone, and even to fatten it
somewhat. This is never the case, however, when the total calorific

' Cf. Lecture V, p. 85.

' The observations concerning the relative loss of weight for the different parts of
the body are as a rule very inadequate and unsatisfactory. We have stated above the
generally accepted view concerning these relations, but we must say that the field has
not yet been well covered. There is no question but that a careful examination of the
losses of the different organs in their various constituents, naturally referred to average
values, will give us a new point of view concerning intermediate metabolism and of the
relations existing between the individual organs. The well-known experiment of
Miescher is the first step in this direction.

' Cf. Lecture XV, p. 3.36.

GENERAL METABOLISM. 637

requirement is met with nitrogen-free food. It is then impossible to pre-
vent the animal from losing weight even when far more fat and carbo-
hydrate are fed to it than corresponds to the calories required when the
dog was in metabohc equilibrium with albumin present in its food.
As soon as the albumin is wanting in the food supply, starvation
metabolism begins, i.e., body albumin begins to be decomposed. It is
true that the animal lives a few days longer than if it were absolutely
star\'ing on account of lack of all food, but it will gradually die as a result
of albumin starvation. The decomposition of albumin shows a peculiar
behavior when var3'ing amounts of albumin are present in the food. The
more albumin the animal eats, the more there is decomposed. To be sure,
by greatly increasing the amount of albumin it is possible for the organism
to store away material, but usually not in the form of albumin itself. The
cells of the body evidently strive to keep the albumin content of the organ-
ism at a constant level. It is possible to bring an animal into so-called
nitrogen equilibrium with different amounts of albumin. Equilibrium is
reached when the organism experimented upon elimmates the same amount
of nitrogen that it receives. This relation is most apparent if instead of
estimating the amount of nitrogen eUminated during a single day, a period
of several days is studied.

The fact that an increase in the albumin income also causes an increase
in the total metabolism apparently helps to enable us to decide whether
the cell-material, or protoplasm, itself takes part directly in the decompo-
sitions and combustions, or whether we have to cUstinguish sharply between
the cell-nutriment and the cell-building-.stones. Here w^ meet with the
most important problem of metal^olism. It is quite generally assumed that
in animal combustions it is chiefly the albumin in the nutriment, also
designated as circulating albumin, which is consumed, while the living
protoplasm is only drawn upon for the outgo when there is a deficiency in
the supply of albumin. There are a number of observations wliich support
this assumption. Above all, it is remarkable how quickly the albumin is
oxidized after its introduction into the organism. Within a few hours the
total amount of nitrogen reappears in the urine. It is hardly absorbed
before its elimination begins. Although the presence of fat and carbo-
hydrate in the food somewhat diminishes the decomposition of albumin,
still on the whole the decomposition of the albumin is about the same as
with a purely meat diet. The fact that the extent of albumin decomposi-
tion is, within wide limits, independent of the albumin content of the body
itself, has also been cited as supporting the above conception. The
assumption that the tissue-cells of the fully developed animal organism
are in a relatively stable condition, and work essentially by means of the
energy obtained from the food, is an attractive one. According to this,
the cell takes up from the blood, or the plasma, the substances which it

638 LECTURE XXVII.

requires for the exercise of its functions. It retains its own cell-material.
The fact that now and then a few cells are broken down and renewed,
hardly affects the metabolism as a whole.

This very simple representation of metabolism is, however, as we shall
find on closer examination, not entirely in accordance with certain facts.
As we have already repeatedly stated, it has become recognized during the
last few years that the cUgestion of the food is not for the sole purpose of
making it capable of absorption. Unquestionably, one of the principal
objects is to make the material which is obtained from different sources
conform to the material out of which the body is made up. The substances
contained in the food are, by means of the various ferments, not only made
capable of absorption, but of assimilation as well. Starch, which is the
glycogen of plants, is broken down into molecules of d-glucose, only to be
changed later into animal glycogen. We do not yet know how much of
the absorbed glucose is changed into the latter compound, nor whether
the glycogen throughout the entire animal kingdom is all of the same nature,
or whether perhaps there are not different kinds of glycogen corresponding
to the different species of animals. Here we cannot decide definitely
whether the preliminary preparation is all-important as regards assimilation,
or whether it merely serves the purpose of making the material capable of
absorption. The question is similar in the case of the fats. In this case
one might even get the impression that the nutriment is deposited in an
unchanged condition. The fat is split by the digestive ferments into its
constituents, fatty acids and glycerol, which, however, unite again in the
intestinal wall. It has been found possible to cause foreign fat to be
deposited in the body. We have already shown that the fat stores occupy
a peculiar position in animal economy. It is a question whether the animal
organism can also utilize substances foreign to it for physiological functions
in cell-metabolism. At the same time we may quite safely assume that
in the case of the fats, digestion only serves to prepare it for absorption. To
be sure, we must confess that the fat stores of different animals under
normal conditions are not homogeneous as regards their composition.
How far these differences in chemical composition depend upon differences
in the nature of the food, remains an open question. In general we may
indeed assume that the fat contained in the stores has a specific composi-
tion for every animal, in case the animal is not deprived of the power to
construct its own fat by decomposition and selection, on account of being
limited to fat of a definite kind.

At all events, it is very remarkable how quickly and easily the animal
organism decomposes, by means of the ferments, the fats and complicated
carbohydrates into their simple components, only to rapidly reconstruct
them again, and eventually, at the time they are consumed, carry away
the resulting products equally rapidly. These very complicated processes

GENERAL METABOLISM. 639

become still more remarkable when we follow, as we have done, the behavior
of albumin in the animal organism. We find that in the alimentary canal
it undergoes a far-reaching decomposition. Now the different albumins
are very similarly constituted in respect to the amino acids which they
contain. With few exceptions they all contain the same building-stones.
Their chief difference Ues in the relative amounts which they contain of
these acids. In spite of these differences the serum albumins are, as far
as our knowledge goes, of the same composition, no matter whether the
albumins contained in the food are closely related to the serum bodies
in their composition or not.' Although the researches in this direction
have only just begun, still a number of observations indicate that the
albumin in the food is so transformed before it reaches the circulation and
the tissues that it loses its original character and becomes of a nature
corresponding to the body albumins, first of all to the serum albumins.
It is, in fact, not the albumins of the food that circulate in the blood and
tissues, but rather those of the body itself.^ Possibly, if we were able to
trace more closely the transformations of the albumins on their path of
absorption and assimilation, and had a better understanding of the albu-
min molecule, the decompositions would then appear to be very simple
ones. At present it appears as if we could not neglect the above-described
relations in the conception of albumin metabolism. Accoi'ding to them,
the decomposition which albumin undergoes does not appear to be a rela-
tively simple process. It is certain that the albumin before it is consumed
must be decomposed again in the tissues into simpler components. The
great question is only with regard to the reason for the changes in the
albumin in the food, so that it becomes like the albumin of the body, when
it is to be consumed so quickly.

It seems to us as if the answer to this question, as we have previously
stated,' will give the solution to the enigma of the large albumin require-
ment. To be sure, the animal organism effects all these changes chiefly
in order that nourishment may be offered to the cells in a form such
that they are able to utilize it. We must always remember that the cells
eventually accomplish their work by means of ferments, and that these are
regulated very sensitively so that they will react only with certain definitely
constituted compounds. Undoubtedly the tissue-cells cannot break down
and utilize starch, or the fat and albumin of the food. The intestine
works over these materials and gives to the cells nutriment of quite specific
composition. The cells are capable of utilizing these definite products and
only these. Their whole construction corresponds to these compounds.

' Emil Abderhalden and Franz Samuely: Z. physiol. Chem. 46, 193 (1905).
' In Lecture XXIX we shall discuss a biological method for determining whether
the albumin nutriment passes directly into the blood and lymph circulation.
' Cf. Lecture XI, p. 223 et seq.

640 LECTURE XXVII.

This is a satisfactory explanation as regards the carbohydrates and fats,
which, at least with fully developed organisms, can, under certain con-
ditions, be regarded solely as combustible materials. The amount of these
substances consumed is determined by the amount of energy which is trans-
formed in the body. If the supply of carbohydrate or fat is greater than
the organism requires at a given time, some of it is laid aside. It is quite
different with albumin. The amount at hand regulates the entire metab-
olism. The more there is present, the greater the metabolism. Why is
so much albumin required? The distinction between circulating albumin,
i.e. albumin which is to be used as fuel, and organized albumin, or that
which is used for the construction of cell-material, does not help us here.'
As a matter of fact, there is no proof that there is any justification for a
sharp distinction between these two kinds of albumin. As we have pre-
viously stated, we prefer to consider the total albumin metabolism from a
single standpoint and to trace the high requirement of albumin on the part
of the animal organism to the fact that in the adaptation of the albumin
in the food to the form required by the body there are quite a number of
the .simpler constituents which are not suitable for synthesizing the new
albumin, and these are eliminated. The intestine contains a mixture of
different amino acids from which it selects those which suit it and in quite
definite porportions. Here again the Law of -the Minimum holds. The
amount of amino acid which is utilized in the synthesis of new albumin is
governed by the amount of that amino acid which is present to the slightest
extent relatively. This conception holds only while we have no positive
proof that the animal cell is capable of forming one amino acid from another
to any considerable extent. For the present our knowledge of the rela-
tions concerning the decompositions in the tissues makes any such assump-
tion appear improbable. Now the most widely cUfferent cell complexes
likewise possess their own characteristic albumin. We refer, for example,
to the histones, which differ again from the albumin of their nutriment,
in this case the serum albumin. Here again the nutriment is broken down,
and again the cell chooses those building-stones which it can utihze, and
rejects the others. Thus we can imagine that in this reconstruction of the
cells, which, although not very extensive of itself, is nevertheless constantly
taking place, a considerable supply of albumin is required. When the
albumin supply is large, the cell is assured of a sufhcient supply of all the
most varied building-stones. The unutilized amino acids are at once
robbed of their amino group, and perhaps the nitrogen-free chains are
further utilized. Even in starvation metabohsm the consumption of
albumin must be remarkably high, for, in this case also, the albumin trans-

' Cf. Emil Abderhalden : Zentr. gesamte Physiol, u. Pathol, des Stoflwechsels, N. F.
1 (1906).

GENERAL METABOLISM. 641

ported from organs of minor importance cannot be utilized directly. Here
again there is a preliminary decomposition and selection of material.

Now the only other striking phenomenon is that an increased supply of
albumin increases the extent of the entire metabolism; i.e., not that of
albumin alone. Perhaps this discovery may be accounted for by the fact
that the animal organism evidently has no depot for storing up the excess
of albumin. This is evident because it is so difficult under normal condi-
tions of nutrition to cause a deposition of albumin in-the fully developed
organism. Under these circumstances it is perfectly conceivable that when
there is an increased supply of albumin there is a greater amount of cellular
transformations so that the other materials are also required.

In this explanation we wish to call particular attention to the importance
that is attached to the maintenance of the perfect h' specific cell construction
in the case of each species of animals and perhaps of every single individual.
Here unquestionably the proteins play the most important part by virtue
of the fact that they offer such a variety of forms. The abundant supply of
albumin guarantees to the animal organism its own individuality and that
of its cells as well as its own metabolism.

We must at this place once more mention the fact that albumin
metabolism has been studied almost entirely from a single point of view.
The rapichty of the albumin decomposition has been identified with the
rapidity of the nitrogen elimination. We have, however, no precise reason
for assuming that the splitting-off of the nitrogen is, as a matter of fact,
the signal for the disruption of the entire molecule. After the formation
of urea, carbon chains remain which may be utilized in a number of different
ways. It is possible that the cells prefer to have so much albumin because
it provides them with all the different materials which they require. It is
perfectly thinkable that these carbon chains can be used to form sugars,
or they might equally well be utilized for the production of fat.

It is, as the above discussion shows, perfectly impossible to give an
explanation of albumin metabolism which shall be based upon exact
experimentation. We can indeed formulate hypotheses, but for the
present there is no preference to be given to any particular one. None of
the present hypotheses satisfactorily unites all the known facts, in such a
way that in every respect all the results of experimentation are clearly
accounted for. We must leave these questions entirely open, and suggest
that new theories and new experiments can alone cause progress, and con-
sequently a discussion of the various attempts at explanation would be
scarcely worth our while. Again and again in the questions arising from
all sorts of different kinds of metabolism we run against the metabolism of
the cell, and cell activity. The conception of intermediary metabolism is
a very accessible one. We are constantly coming in contact with it. It
is here the place to state that contrary to what one might expect by reading

642 LECTURE XXVII.

the literature, as far as we are concerned it is an unknown quantity, and
at present we have practically no clear insight into cell-metabolism. For
the time being we recognize only the general stages of metabolism as a
whole. There is here a vast field for experimentation which has scarcely
been touched upon. It is only by a clear recognition of this fact that it
will be possible for us to penetrate fully unprejudiced into this obscurity,
and with new methods and new resources succeed in gradually developing
more and more fa(rts which will replace the hypotheses. We have gone
into albumin metabolism upon a somewhat broad basis, because eventually
all questions concerning metabolism, no matter what their nature may be,
directly or indirectly penetrate into the problem of albumin metabolism
in the animal organism. The uncertainty which at present envelops the
latter to some extent affects all other investigations in this field, and
explains, at least to some extent, the different answers which have been
given the apparently similar questions concerning metabolism.

In this connection we must once more remember that it is exceedingly
difficult to cause a deposition of albumin in the animal organism. Muscular
work has a remarkably favorable action in this direction. The significance
of work for accomplishing an albumin "fattening" has been recognized
by the physician. A retention of nitrogen has alone been satisfactorily
established in this connection. Less nitrogen appears in the urine than
the organism receives. We do not know what becomes of this nitrogen
that remains in the body. From the experiments of Schreuer' we know
that the "albumin" retained in the body is not equivalent to the remaining
albumin in the body. The deposited albumin is readily lost again, for when
the ordinary diet is resorted to, the organism soon returns to its usual
albumin condition. This is true especially of the nitrogen retention which
is brought about by a large supply of albumin. If, at the same time,
demands are placed upon a function of the body, e.g., that of the muscles,
we can easily imagine that the individual cells will attempt to utilize this
for increasing their cell material.

In the fully developed organism there is also some occasion for albumin
being retained. It requires constructive material. Direct experiments
teach that the developed organism in fact constantly acquires nitrogen.
The adult organism during pregnancy finds itself placed under quite similar
conditions as during youth. Here again there is a constant formation of
new tissue to an unusual extent. Corresponding to this, P. Bar and R.
Daunay^ showed that in the case of a gravid bitch, nitrogen was constantly
held back from the nourishment. This nitrogen retention was also notice-
able when the food was the same as that with which the animal was in

Pfliiger's Arch. 110, 227 (1905). Cf. Karl Bornstein: ibid. 106, 66 (1904).
J. physiol. et pathol. general, 1, 832 (1905).

GENERAL METABOLISM. 643

nitrogen equilibrium before the period of gestation. From this fact it
follows that the developing fetus does not live at the expense of the organ-
ism of the mother, but to a certain extent is included in the general nourish-
ment. The organism of the pregnant mother utihzes to better advantage
than usual the substances contained in the food and especially the protein.

In discussing the replacement of one foodstuff by another and the posi-
tion of each in animal economy, we have considered the suitability of each
kind of food for definite functions. We have seen that the carbohydrate
decomposition is proportional to the amount of muscular work, and that
fats and also albumins can replace carbohydrates as sources of energy.'

It remains for us to mention briefly the influence of certain external
conditions upon metabolism. Above all, the influence of the surround-
ings is of interest. Up to the present time the effect of temperature has
alone been studied to any extent. In this respect the poikilothermous
animals are very different from the homothermous ones. In the former,
metabolism runs parallel to the variations in temperature, i.e., it decreases
with a fall in the external temperature and increases with a rise in tem-
perature. This can be demonstrated very clearly by studying the respi-
ratory exchange. The warm-blooded animals, on the other hand,
behave quite differently. With them metabolism increases with falling
temperature and decreases with rising temperature. This is due to the
fact that the warm-blooded animals seek to keep their body-temperature
the same, irrespective of the external conditions. The loss of heat caused
by a fall in the external temperature is compensated by an increased
metabolism. The muscles are the principal seat of this change in the extent
of the metabolism. If the muscular activity which may be expressed
by movements, by shivering, or also by muscular tensions, is prevented
by curare poisoning or by severing the spinal cord high up, the heat
regulation ceases. The principle that the metabohsm of homothermous
animals increases with falling temperature and conversely cUminishes with
rising temperature holds only in part. It has been shown that a consid-
erable rise of temperature also has a similar effect upon the warm-blooded
animals as upon the cold-blooded ones; the metabolism increases so that
there is an increased production of heat against which the organism seeks
to protect itself by giving off more heat. The chief difference, then,
between the homothermous animals and the poikilothermous ones is their
opposite behavior with regard to a fall in temperature.

' There are a great many experiments concerning metabolism under varying condi-
tions. We cannot consider them here, because in most cases it is difficult to establish
the exact effect of the different factors. We would refer to the influence of high altitude
upon metabolism, and of a sea-shore climate. Cf. N. Zuntz, A. Loewy, F. Miiller, and
W. Caspari: Hohenkliraa und Bergwanderungen in ihrer Wirkung auf den Menschen.
Bong & Cie, 1906. A. Jaquet and R. Stahelin: Arch, exper. Path. Pharm. 46, 274
(1901). Loewy and Miiller: Pfiiiger's Arch. 103, 1 (1904).

LECTURE XXVIII.

GENERAL METABOLISM.

II.

The human and animal organism requires for tlie maintenance of its
bodily condition and for the exercise of its functions a perfectly definite
amount of nutriment. The nutritional requirement depends, naturally,
upon various external conditions. Unquestionably individual peculiari-
ties also come into play here, at least to some extent. One very important
factor is the amount of work to be performed. It is also self-evident that
the large amount of new tissue which is being formed in the growing
organism also influences the amount of food required.

There are a numl^er of different ways for getting an idea of the amount
of food required under definite conditions. For one thing we can ascertain
the diet chosen by different people of various caUings, and estimate from
its composition the calorific value, and use this as a basis. If this is done
with a number of different individuals for each class, then we shall obtain
very useful average values. A single observation does not give a reliable
indication of the food requirement. Certain circumstances, such as the
nature of the food chosen, its utilization, especially in individual cases,
lessen the value of the calculated amount of calories from a single observa-
tion. The greater the amount of material worked over, and the more
uniform the external conditions are, the less influence will be exerted by
individual peculiarities. It is perfectly clear that all such estimations
will involve more or less error, and at best we can only arrive at approxi-
mations.

It is usually not possible to determine in such investigations the extent to
which the food material is utilized. Such computations must be based upon
exact experiments on metabolism. Nevertheless, such observations have
great value from a hygienic-sociological standpoint. The phj'siology of
metabolism has become epoch-making in this direction. By means of it,
attention has been called to the altogether insufficient nourishment of
certain classes. It is perfectly clear that a permanent underfeeding and
too low standard of life must eventually tend to weaken the individual. _
The resistance towards injurious external influences, towards infectious
diseases, etc., becomes lessened, morbidity and mortality increase, the
growth of children becomes retarded, the number of able-bodied men for

644

GENERAL METABOLISM.

645

the army becomes smaller, and before long the inadequate nourishment
of a class of people casts its shadow in many directions.'

Carbohy-
drate.

Laborer at moderate work

Laborer at hard work

Well-paid craftsman

Cabinet-maker (40 years old)

Street porter (36 years old)

Young physician, Munich

Lawyer, Munich

Carpenters, coopers, and locksmiths in Ba-
varia

University professor, Munich

Bavarian woodsmen

Brewery man at hard work

Peasant man

German laborer (mean from 11 families) . .

Miners at hard work

Weavers' families in Saxony

Two laborers' families in Frankfurt a. M . .

Laborer in Berlin

Italian brickmaker

French laborer

English laborer

Scandinavian laborer

Well-nourished tailor, England

Hard-working weaver

Hard-working blacksmith

Seamstresses, London

Students, Japan

Salesman

Swedish laborer at moderate work . . . .

Swedish laborer at hard work

Transylvanian farm-hand

Factory people in Central Russia:

Men and women

Boys

Country folk near Moscow:

Men

Boys

Fishermen on the Wolga:

Men

Women

118
137
151
131
133
127
80

122
100
135
190
143

72
133

65

167
138
140
198
131
151
176
54
83
55
134
189
182

129
102

319

219

56
173
54

34

100

208
73

108
49

113
49
49
69

117
80
34

109
39
43
71
29
14
6
79

101
93

80
51

33

28

57
43

500
352
479
494
422
362
222

570
240
876
599
788
451
634
485
419
490
675
502
435
710
525
622
667
292
622
394
485
673
968

584
487

589
471

563

3091
3678
3148
3242
3214
2890
2437

3206
2373
6091
3993
4848
2608
4240
2710
2424
3075
4605
3419
2733
4811
3096
3618
4179
1699
3019
1898
3322
4545
5217

3708
2896

3236
2637

4369
2909

' Cf. Paul Mombert: Das Nahrungswesen. Gustav Fischer. Jena, 1904. Here unfor-
tunately we can but touch upon these problems which are so very important as regards
the common people. It is highly important that these relations should be studied
closely.

' These values should be at least 8 per cent lower to correspond to the calories actually
utilized by the organism. A part of the food is not absorbed. This amount varies with
different food, as we have seen. The results given are comparable without making this
deduction. For accurate data it would be necessary to know the amount utilized from
case to case. The above table is from a summary in J. Konig's " Die menschliche
Nah rungs- und Genussmittel, ihrer Herstellung, Zusammensetzung und Beschaffenheit,"
Julius Springer, Berlin, 4th edition, Vol. 1, p. 388 (1904).

646

LECTURE XXVIII.

In the table on page 645 are given the results of a few observations
with regard to the amount of nourishment taken by different classes of
people.

Atwater has published the following values for the different classes in
the United States.'

Food Pur-
chased.

Food Eaten.

Digestible Nu-
trients in Food
Eaten.

Fuel Value in
Calories.

Class and Occupation.

c .

c i
■I S

o O
&.

101
113

110
127
109
180
200

i

5

c

128
153

136
181
102
365
304

1 ^

Is

o ^

r-

c .

ii

r

97
106

107
106
108

E
g
O
c

1

121
142

129
146
100

P

465
406

437
363
432

B tfi

c £

o U

88
97

99
98
99

1

5

c

117
137

124
141
96

1 ^

Si

o

453
395

426
354
422

1

3560
3605

3530
3880
3175
8850
6905

5740
4462

3975
2910

3530
3465

3826

3705
3405

»

3435
3420

3430
3305
3145

4335

3275
2780

3430
3280

3524

5

Farmers' Families (9) ^ . .
Mechanics' Families (9) .
Professional Men's Fami-
lies (9)

College Students' Clubs (5)
Laborer's Family ....
Mason . . i at hardest

476
420

442
402
434
1150
365

3305
3295

3305
3170
3030

Man in Adirondacks in mid-

200

216

367

190

209

358

4190

Football Player

Sandow, "The Strong Man "
Teachers' Families:

Illinois

Indiana

Officials' Families with lit-
tle work:

Connecticut

Pennsylvania

Mechanics' Families with
little work (5) . . . .
Students' Clubs:

Young Men (16) ... .

Young Ladies (4) ...

181
244

124
111

110

98

114

105
101

292
151

158
110

136
155

170

147
139

557
502

487
349

442
396

436

465
414

101
106

107
91

105

113
102

129
145

154

441
340

437
380

407

C. Voit and Max Rubner' compute the total food consumption per day
for adults to be per head:

' W. O. Atwater: Storr's Agricul. Experim. Station, Storr's Conn. Ann. Report, 9, 152
(1896) (1891-1896).

' The numbers in parenthesis represent the number of dietary studies upon which
these average values are based. In several cases experiments were made with a family
in the fall and spring of the year, thus making two dietary studies with one family. Thus
in the first case there were five farmers' families which were under observation. (Trans-
lator.)

' Cf. M. Rubner in " Handbuch der Emahrungstherapie und Diatetic," by E. von
Leyden, Part 1, 154 (1857), and Konig: loc cit.

GENERAL METABOLISM.

647

Place.

Protein.

Fat.

Carbohy-
drates.

Calories.

Grams.
84
96
98
98

Grams.
31
65
64
60

Grams.
414
492
465
416

2350

3036

Paris

2929

2696

From the values published by Atwater it is apparent that the diet
chosen by people of various callings is very different both as regards the
composition and calorific value. In order to be able to estimate the extent
to which the food chosen suffices to meet the requirements which are placed
upon the organism, it is necessary to make use of the average values
obtained by direct experiments upon metabolism. In order to establish
the minimum requirement of the human organism, the metabolism is
studied under conditions at which the muscles are as much at rest as possi-
ble. In twenty-four hours the requirement per kilogram of body-weight
has been found to amount to from 30 to 36 calories. The average man
may be assumed to weigh about 70 kilograms, and on this basis the mini-
mum requirement per twenty-four hours would be 2100 to 2520 calories.
This value does not hold for absolute rest, when the requirement is con-
siderably less. The resting organism is satisfied with considerably less
food, as has been shown by metabolism experiments with a woman in
hysterical coma.' The minimum requirement was then only 1680 calories.
It is impossible for the muscles to be in such a state of rest when the per-
son is awake. Now from the computed values for a person resting as
much as possible, we may pass judgment upon the values given in the
above tables. Unquestionably some of the above individuals were under-
fed. According to the wide experience of Voit, the daily diet of an adult
engaged during nine or ten hours in ordinary work should be about 2749
calories.^ This amount may be distributed among the different nutrients
as follows: Protein 118 grams, fat 56.0 grams, carbohydrates 500 grams.
The more work there is to be done, the more calories are needed. Thus
Voit estimates that soldiers engaged in maneuvers require 135 grams
albumin, 80 grams fat, and 500 grams carbohydrates, amounting to 3018
calories. In time of war, however, the requirement he states to be 145
grams protein, 100 grams fat, and 500 grams carbohydrates, which is
equivalent to 3218 calories. Working women, on the other hand, require
but 2200 calories, which should be composed of 94 grams protein, 45 grams
fat, and 490 grams carbohydrates.

' Sond& and Tigerstedt: Skand. Arch. Physiol. 6, 1 (1895). J. E. Johansson, E.
Landgren, K. Sond^n and R. Tigerstedt: ibid. 7, 29 (1896).

' After subtracting the calories lost by insufficient utilization. This deduction is
not made in the case of the values given for the separate nutrients.

648 LECTURE XXVIII.

In order to get a better idea of what these values mean, we will state
that '

100 grams of protein are contained in
5000 grams potatoes, 650 grams fat pork,

4200 grams human milk, 620 grams yolk of hens' eggs,

3000 grams cabbage, 600 grams fat beef,

3000 grams cow's milk, 500 grams lean beef,

1250 grams rice, 430 grams peas.

800 grams wheat.
For each 100 grams of protein there is pre.sent

Carbohydrates.

Fat.

Cow's milk

Peas

140
230
270

580
990
1000

107
21

Human milk

Wheat

170
14

11

Potatoes

0

There has been much discussion as to whether the amounts of abumin
given above as representing the average albumin requirement are too high
or not. It has been found possible under certain conditions to get along
with less protein. It is a question, however, whether these results
obtained in single experiments, which, moreover, cover as a rule but a brief
period of time, should be used for determining the ordinary requirement.
Aside from individual peculiarities which unquestionably come into play
here, there are certain external conditions which are to be considered as
exerting an influence in a way that it is difficult to define accurately.
The food requirement is different with different nations according to the
climate in which they live. It is certain that habits also play a part.
The human and animal organism must also be independent, within certain
narrow limits, of the amount of protein in the food. The amount of
protein which the organism receives from day to day changes considerably
when the diet is a varied one. The amount of non-nitrogenous material
which the organism receives, and which is almost invariably sufficient in
quantity, enables the organism to be satisfied with as little as 100 or even
90 grams of protein per day. It would of course be wrong to base a definite
standard of the food requirement upon the investigations which have been
made up to the present time. It would be possible to formulate this
requirement more sharply if we knew more precisely the conditions under
which the values were obtained in different cases. Even the body-weight
as such is not an exact factor. A very muscular individual will require

' These values are taken from Konig's " Die menschliche Nahrung- und Genussmittel,
etc." They refer to the food in its natural condition.

GENERAL METABOLISM. 649

more protein than a bony person with but slight muscular development.
On the other hand, we are at present unable to measure the actual amount
of work done by people of different callings, which would give us exact
data for the calorific requirements. At all events, the protein content of
the food is practically of chief importance, as regards a definite food
requirement. From a practical standpoint, furthermore, it is hardly
desirable to have a definite minimum established as regards the amount
of protein required by the organism. If it should be attempted to
make the food contain such an amount, it would be very easy to fall
below it from time to time, and this would lead to albumin losses from
the body. There is absolutely no reason, aside from the expense, of
being afraid that too much albumin will be eaten. The body ea.sily
assumes a state of equilibrium with a larger .supply of protein. We have
seen that it is very difficult indeed to cause an accumulation of albumin
in the body.

The question of expense is the most important factor as regards the best
way to meet the calorific requirements with non-nitrogenous food after
the organism has received sufficient albumin. Carbohydrates are cheapest.
The amount of these in the above-mentioned chets is also considerable.
They can be easily taken care of by the intestine. Fat, unfortunately, is
very expensive, although, to be sure, an equal weight of it has more than
twice as much calorific value as the carbohydrates.

In this connection we must call attention to a peculiarity. When persons
are obliged to resort to a definite diet which is about the same from day to
day, they often lose in weight, and show even in their outward appearance
that they are not v/ell-nourished, even although there may have been enough
calories in the food. We can to some extent understand that a diet which
is absolutely non-irritating, and remains exactly the same from day to day,
will not be as nutritious as one of different composition which may not
have any greater calorific value. We have seen that the smell, taste, and
other sensations play an important part in the preparation of the digestive
fluids. Now when the sensation is exactly the same from day to day,
and the food is free from irritating substances, it soon fails to cause
any stimulation of these sensations. The way in which the food is pre-
pared also exerts an important effect. This governs largely the extent to
which the smell and taste nerves are stimulated, and moreover provides
for a greater utilization of the food material.

One question which has been considerably discussed is whether the
human organism should obtain its food preferably from the vegetable
kingdom exclusively, or whether a mixed diet is preferable.' Certain

' Cf. G. von Bunge: Der Vegetarianismus, 2d edition. A. Hirschwald Berlin, 1901.
Ferdinand Hueppe; Der moderne Vegetarianismus. A. Hirschwald, Berlin, 1900. W.
Caspari: Physiologische Studien ijber Vegetarianismus. Martin Hager, Bonn, 1905.

650

LECTURE XXVIII.

anatomical observations apparently indicate that man is adjusted to take
care of a diet containing both meat and vegetables. The intestine is
neither as short as in the case of the pure carnivora, nor as long as that of
the herbivora. It is interesting to find that nations, such as the Chinese
and Japanese for example, which are accustomed to a diet in which vege-
tables predominate, have a longer intestine than individuals of a nation
which is accustomed to a meat diet. As regards the shape of the teeth,
it is not possible to draw any definite conclusions; and it is also true as
regards the historical development of the human race, that there is no proof
that man was originally accustomed to a vegetable diet. Our knowledge
of cookery has to a certain extent made us independent of the nature of
the raw material. This is particularly so with regard to the products of
the vegetable kingdom. Cooking enables us to get at the contents of the
plant cells better, which were originally enveloped by cellulose; and
important foods, such as the potato, are made more accessible to the action
of diastase. If we really wish to make a definite decision with regard to
the relative values of vegetables and meats, we must, in the first place,
compare the extents to which each is utilized. This may be ascertained
by the analysis of the fasces, determining the amount of unabsorbed
material. Certain factors come into play here which make it hard for us
to decide. A great deal depends upon the nature of the food. A diet rich
in starch may have an unfavorable action upon the absorption of the other
nutriment, on account of the fermentation processes resulting and the
formation of acid (butyric acid), which accelerates the peristalsis of the
intestines, thereby causing a prompt evacuation. Food rich in cellulose
will have the same effect. Individual peculiarities also undoubtedly come
into play here. At the same time it is perfectly possible for us to obtain
approximate values for the extent to which the various foodstuffs are
utihzed in the human organism. Such values are given in the table on
the following page.

The more incomplete utilization of the protein in vegetables as com-
pared to that of flesh foods is also shown by the results of experiments by
Atwater and Langworth ' with vegetable, meat, and mixed diets. This is
shown in the following summary.

Experi-
ment
No.

Nitrogen in Grams per Day.

Food.

In Food.

In Urine.

In Fceces.

Nitrogen
unutilized.

Purely vegetable

Mixed ( Average amount of meat,
diet I Large amount of meat .

55
74
56

13.8
19.4
33.1

13.9
15.6
24.5

3.9
2.4
2.9

28.3%
12.6%

8.9%

A Digest of Metabolism Experiments, Washington, 1897.

GENERAL METABOLISM.

651

1. ANIMAL FOODS.

Food Absorbed in Per cents of that Eaten.

Dry Sub-
stance.

Milk —

Children

Adults

Cheese

Eggs

Flesh —

From slaughtered animals

From fish

Slaughter-house scraps . .
Fat —

Butter

Oleomargarine

Lard

96.0
94.5
92.0
95.0

95.5
95.0
90.0

Nitro-

genous

Fat.

Substance.

95.5

97.0

93.5

95.0

95.0

90.0

97.0

95.0

97.5

94.0

97.0

91.0

89.0

92.0

97.0

96.5

96.0

Carbohy-
drate.

2. VEGETABLES.

Wheat flour or wheat bread

Fine

Medium

Coarse

Rye meal or rye flour —

Fine

Medium

Coarse

Rice

Indian meal

Whole as meal —

Legumes

Peas, beans

Potatoes

Green vegetables

Mushrooms

Cocoa

95.0
93.5
90.0

93.0
88.5
84.0
96.0
93.5

81.5
90.5
93.0
82.0
80.0

81.0

75.0

98.5

75.0

60.0

97.5

72.0

55.0

92.5

73.0

95.8

68.0

93.3

60.0

90.0

80.0

93.0

99.0

83.0

70.0

96.5

70.0

30.0

84.5

84.5

40.0

95.0

78.0

97.5

95.8

72.0

93.0

83.5

70.0

41.5

94.5

98.0

3. MIXED DIET.

Largely animal

Largely vegetable ....

Average diet

The same with white bread
The same with rye bread .

95.0
90.0
94.0
95.0
91.0

91.0

95.0

97.0

78.0

86.0

93.0

85.0

92.0

95.0

88.0

92.0

96.0

82.0

92.0

93.0

Metabolism experiments with purely vegetable diets show that it is per-
fectly possible to nourish a youthful, vigorous organism with vegetables

* KSnig: loc- cU.

652 LECTURE XXVIII.

alone and to maintain it at tlie highest stage of bodily and mental vigor.'
A diet consisting entirely of vegetables is inadvisable for the following
reasons: In the first place, vegetables are not utilized very advantageously,
as the above tables show; this is particularly true of the protein which they
contain. It must be stated, however, in this connection, that the values
in the tables were determined solely by the amount of nitrogen in the food.
This is not quite right, for meat contains nitrogenous extractive substances
which are not of an albuminous nature. For this reason the values given
for the protein in the meat were a little too high. This, however, does not
materially influence the comparison. A vegetable diet has the further
disadvantage that it lacks savor. To be sure, this may be remedied by
artificial additions, and by exercising especial care in the preparation of
the food. A vegetable diet is especially objectionable on account of the
greater volume of the food.

All our present knowledge, both from the standpoint of experiments on
metabolism and practical e.xperience, justify us in assuming that a mixed
diet is to be preferred as food for a people. There is no reason why we
should attempt to eliminate animal food from our rations.

It has never been positively proved that a flesh diet, even when it pre-
ponderates, is harmful. All statements with regard to the injurious effects
of a meat diet are based upon indirect conclusions, which are capable of
two interpretations. We must admit that the human organism is capable
of deriving sufficient nourishment from a vegetable diet, if it is provided in
sufficient quantity. It does not seem true, from the above experiments,
that the organism accustoms itself to vegetable food in the sense that the
vegetable material is consumed to better advantage after a time. It would
not be at all advisable, on the other hand, to restrict the diet for any length
of time to meat, and chiefly because of the fact that there is then a lack
of material which tends to promote the peristalsis of the intestines. In
the case of the carnivora, the same effect as that produced by cellulose is
obtained from the fragments of bone and other difficultly-digestible material
which the animal swallows with its food.

The whole question concerning the relative advantages of vegetable,
meat, or mixed diets rests largely upon one important point, namely, which
kinds of material are utilized in the body to the best advantage. We have
again and again stated that the food does not, under normal conditions,
become part of our bodies in the form that it is eaten, but it is 'the con-
stituents which result from a complete disintegration of the food that are
suitable for the body. All nourishment is eventually assimilated in our
tissues. If we hold to the standpoint that the tissue cells — of course in
a restricted sense — are quite independent of the nature of the food which

' Cf. Caspar! ; loc. cit. p. 122.

GENERAL METABOLISM.

653

is eaten, and are affected solely by the nutriment which they receive from
the circulation, and which has already been assimilated, then we can formu-
late the whole question regarding the value of animal or vegetable food in
such a way that we shall have to know merely which of the two contains
the building-stones of protein in the proportions corresponding more closely
to the albumins of the body. As regards the assimilation of the albumin-
ous substances in the animal organism, the first thing to consider is whether
the protein introduced can be decomposed into its constituents by means
of the ferments contained in the intestine, and then whether these build-
ing-stones are present in the right proportions. If we examine the pro-
teins contained in vegetable and animal food from this point of view, we
shall find that the latter correspond more closely to the composition of the
protein contained in our own tissues. This is shown particularly plainly
in the case of glutamic acid, as is shown by the values given in the follow-
ing table: '

One hundred grams of the protein contain of glutamic acid in grams:

Gliadin from wheat .
Gliadin from rye . .
Hordein from barley
Zein from Indian meal

Glutein

Legumin

Vignin from peas . .

Conglutin from Lupinus luteus

Casein

Egg-albumin

Albumin from fish-muscle . .
Albumin from ox-muscle . .

Serum-albumin

Serum-globulin

20.96
10.7
8.0
8.9
11.9
7.7
8.5

Unquestionably our present knowledge indicates that the albumin from
vegetable foods gives rise to more waste products in the intestine than that
from flesh foods. On the other hand, it is perfectly conceivable that a mix-
ture of animal and vegetable proteins would enable the animal organism
to utilize certain constituents of the latter in common with the former
which might otherwise be worthless perhaps on account of a deficiency in
glutamic acid. This is, at present, merely a suggestion. It is well, how-
ever, to consider such questions from all possible points of view.

The fact that vegetarians are often under-nourished is worthy of men-
tion. They are then obliged to draw upon their own albumin, and live,
so to speak, upon animal protein. They are then false to their own
doctrine !

In order to determine the nutritive value of a foodstuff, we must in the
first place know its composition. In the following table the composition of
one of the most important foods is given. In infancy milk is the chief, if
not the only, form of nourishment. We have already discussed the compo-
sition of its ash, and have found that it is characteristic of the milk of every

' Cf. Lecture IX, p. 172 et seq., and T. B. Osborne and R. D. Gilbert: Am. J. Physiol.
15, 303 (1906).

654

LECTURE XXVIII.

species of animals. This is also true of the organic constituents, especially
protein, fat and carbohydrate, as the following table shows:
One hundred parts by weight of milk contain: '

Specie:

Dog I . . . .
Dog II ... .

Pig I

Pig II . . . .
Pig III . . . .

Sheep

Goat

Guinea pig I .
Guinea pig II .
Rabbit ....

Cat I

Cat II

Cat III . . . .
Cat IV . . . .

Cow

Buffalo ' . . .
Zebra' . . . .
Camel ' . . . .
Lama ' . . . .
Reindeer ' . .
Horse ' . . . .

Ass '

Mule 2

Elephant ' . .
Caaing-whale '
Hippopotamus '

Casein.

Albumin.

Total
Protein.

Fat.

4.80

2.64

7.44

11.62

4.84

2.43

7.27

12.19

3.76

1.45

5.21

9.54

3.26

1.55

4.81

7.09

3.71 .

1.65

5.36

6.32

4.08

0.80

4.88

9.29

2.91

0.76

3.67

4.33

4.60

0.49

5.09

7.31

4.79

0.61

5.40

6.96

8.17

2.21

10.38

16.71

3.79

3.30

7.09

4.49

3.79

3.11

6.90

4.80

3.69

3.29

6.98

4.98

3.59

3.49

7.08

4.76

2.90

0.50

3.40

3.70

4.26

0.46

4.72

7.51

3.03

4.80

3.49

0.38

3.87

2.87

3.00

0.90

3.90

3.15

8.38

1.51

9.89

17.09

1.30

0.75

2.05

1.14

0.79

1.06

1.85

1.37

2.63

1.92

3.45

20.58

43.76

4.51

5.04
3.61

2.31
2.02
1.98
4.79
4.80
4.71
4.82
4.95
4.77
5.34
5.39
5.60
2.82
5.87
6.19
5.69
7.18

Human milk differs from all of the above varieties e.xeept ass's milk, in
containing more albumin than casein. One hundred parts by weight of
human milk contain:'

Casein.

Albumin.

Total Protein.

Fat.

Milk-sugar.

0.80

1.21

2.01

3.74

6.37

Milk, besides containing casein and albumin, also contains a small
amount of globulin.'' The composition of the milk with a uniform diet
varies but slightly during the nursing period, with the exception of that

■ Cf. Emil Abderhalden: Z. physiol. Chera. 26, 487 (1899); 27, 408 (1899).

' Computed from several analyses. Cf. Konig: loc. cil. pp. 661, 663, and 664.

' Average values from Konig: loc. cit. p. 598.

< Wroblewski [Z. physiol. Chem. 26, 308 (1898-99)] also believes that a different pro-
tein which he calls opalisin is present, but its isolation and description are not very
convincing.

GENERAL METABOLISM.

655

■which flows shortly after birth. This contains far more protein, and is
called the colostrum. Human colostrum contains in 100 parts by weight
in grams:

Nitrogenous
Substances.

Fat.

Milk-sugar.

3.07

3.34

0.40

The colostrum is found in all species of mammals. With the cow the
relations are as follows: 100 parts by weight of milk contain 2.90 grams
casein, 0.50 gram albumin, 3.70 grams fat, and 4.95 grams milk-sugar.
One hundred parts by weight of colostrum contain 4.19 grams casein,
12.99 grams albumin and globulin, 3.97 grams fat, and 2.28 grams milk-
sugar. The exact significance of the colostrum is not known. We can
indeed imagine that the tissues and cells of the new-born, which now exer-
cise certain functions for the first time, require a considerable supply of
protein.

The composition of the milk is dependent upon a number of external
conditions.' Cow's milk, especially, has been much studied as regards
the influence of various factors upon the composition and amount pro-
duced per day. One of the chief factors is the breed, and another the
nature of the nourishment the animal receives. Moving about and work
have an effect.

The variations in the composition of the milk are not great under
normal conditions. This is very important. The fact that the milk of
different species of animals varies greatly is of much significance. The
composition of the milk evidently has an effect upon the rate of devel-
opment of the suckling.^ It is natural to expect that the richer the milk
is in its organic and inorganic constituents, the more rapidly the suckling
is able to build up its tissues. If the milk of different species of animals
all had the same composition, then the desired effect could be produced
only by means of a much greater production of milk, and similarly a corre-
spondingly greater quantity would have to be taken into the system of the
suckling. The question arises whether the milk of one species of animals
can be substituted for that of another. Our experience concerning meta-
boHsm does not show us a priori any reason why this could not be done,
provided of course that the suckling should receive the same amounts of
nutriment, both quahtatively and quantitatively.' It is, to be sure, con-

' Cf. Konig: loc. cit. p. 601.
' Cf. Lecture XVII, p. 404.

' Cf. M;ix Rubner and Otto Heubner: Z. e.xper. Path. Therap. 1 (1905). Franz
Tangl: Pfiiiger's .\rch. 104, 45.3 (1905). Camerer: Z. Biol. 16, 24 (ISSO); 20, 5G6 (1884).

656 LECTURE XXVIII.

ceivable that the albumin of one kind of milk may be differently constituted
from that of another in a quite specific way. In the case of sucklings the
most important function of the food is to build up the cells. Within a
short time the animal doubles its original weight. We can imagine that
some kinds of protein are not suitable for being introduced into the cell.
Such ideas were very well justified at the time when it was assumed that
the protein was decomposed in the intestine only to the peptone stage and
that these products were absorbed, and when there was no evidence at
hand concerning the composition of the different proteins of the body.
After it was ascertained, however, that the suckling was able from the
protein in its food to construct all the different proteins contained in the
various fluids of the body and in the tissues, the composition of which is
quite cUfferent from that of casein,' it hardly seemed right for us to lay too
much stress upon the quantitative composition of the protein in the food.
Even casein is decomposed while it is in the ahmentary canal. Outside
the intestine the various cleavage-products unite in various ways to form
new proteins. Even the albumin contained in milk is made capable of
absorption by means of changes which take place while it is in the intes-
tine. This does not imply by any means that the chemical composition
of the various proteins is entirely a matter of indifference. There are
no grounds for any such a.ssumption. It is also conceivable that the
caseins from different varieties of milk contain certain specific groups. At
present, to be sure, we do not know of any such. All that we do know is
that up to the present time the different kinds of casein which have been
studied all contain the same building-stones and apparently in about the
same quantitative relations.- We would, however, far exceed the present
state of our knowledge if we were to conclude definitely that all the different
varieties are identical because they are composed of the same constituents.
It is perfectly clear that the same amino acids may be combined in a num-
ber of different ways in the complex molecule. The number of possible
isomers is very large. We have already seen in studying fermentations
that very slight differences in the construction of the molecule are of much
biological significance.

The casein of human milk differs from that of cow's milk in that rennin
throws it down in the form of a much finer flock. It is also easier to pre-
cipitate casein from cow's milk by slightly acidifying it with acetic acid.
From human milk it is very difficult to precipitate the casein by means of
acetic acid. At ordinary temperatures the precipitation is at best very
incomplete, and in most cases no precipitate at aU is formed. In order to
throw down completely the casein from human milk, it is necessary to
carefully acidify it slightly, dilute it with water, and keep it at 37° C. for

> Cf. Lecture X, p. 211.

2 Emil Abderhalden and Alfred Schittenhelm : Z. physiol. Chem. 47 (1906).

GENERAL METABOLISM. 657

some time. In spite of this different behavior of the casein from the two
kinds of milk, which may be due to several causes, we are not justified in
assuming that there is any great difference in the nature of the casein.

On tlie other hand, just as we cannot safely assume from our present
chemical knowledge that the composition and nature of the protein from
different kinds of milk are dissimilar, so we are not justified in assuming
that the milk of the different species of animals is quantitatively but not
qualitatively different. The present state of our knowledge concerning
the composition and nature of the different constituents of milk does not
tell us how completely the milk of one species may be replaced by another.
This does not by any means imply that it is impossible to effect a satis-
factory replacement. We only wish to emphasize at this place how far
our present knowledge is from the desired goal, and how far the present
demands and concessions have stretched beyond the boundaries of our
actual knowledge. At present we are obliged to depend almost entirely
upon practical experience which receives but slight support in the ana-
lytical values obtained from the investigation of milk. We must emphasize
the fact that our present methods of examining milk, particularly the
analysis of the ash, show us merely what elements are present and in
what proportions. The presence of sulphuric acid in the ash may be
accounted for in several ways. It may occur in the milk as such, or the
sulphur may be present in some state of combination other than that of
sulphate. On the other hand, the old idea that the intestine is only able
to bring about certain slight changes in the food, which is then absorbed
after having been reconstructed as little as possible, is more and more to
be discarded. As a matter of fact, the changes which take place while
the food is in the intestine are quite considerable. The assimilation begins
in the intestinal canal. The synthetic capabilities of the animal organism
are much greater than was formerly assumed. It is far less dependent
upon the nature of the food which it receives than was once believed to
be the case.

Practical experience has shown that it is not possible to replace entirely
satisfactorily the mother's milk with that of some other species, or by means
of a milk substitute. The mortality of infants nourished at the breast is
much less than that of infants brought up in some other way. It is an
open question, however, whether this increased mortality is wholly due
to insufficient nourishment. In many cases it is perfectly true that the
children of women who are not able to nurse their children, or at least only
for a short time, are in many cases not as strong as the children of normal
women. Statistics in this direction, therefore, to be useful must take into
account not merely whether the child was brought up on mother's milk,
or upon a milk substitute, but it should also be stated why the mother's
milk was abandoned. It is perfectly clear that if a sickly child is placed

658 LECTURE XXVIII.

upon artificial feeding, it will be hard for it to utilize the nutriment to best
advantage. The child has at the start a defective circulation. It is hard
for the weakened cells to carry out a thorough assimilation and trans-
formation of the food materials. They tend to become weaker and weaker,
and lose more and more the ability of reconstructing the material. Numer-
ous complications lessen the value of the artificial nourishment which the
child receives. If milk from an animal is used, large amounts of micro-
organisms are invarialily present which cause unfavorable effects in the
bowels of the child. These organisms may be killed by sterilization, and
then their harmful effects will not be felt, but on the other hand it has been
found that in sterilization changes are produced in the milk itself which
make it more difficultly digestible. Perhaps it serves to "denaturize"
the proteins in the milk, so that it is harder for the ferments to act upon
them. It has been found possible to carry out the sterilization process
in such a way that this injury to the milk itself is reduced to a minimum.
One great danger to be feared in the artificial feeding of infants is the over-
loading of the alimentary canal. Under normal conditions the infant has
to work pretty hard to get its food. In sucking out the milk the child
becomes tired, so that after a time it stops feeding.

Social relations undoubtedly exert a great influence upon the prevailing
conditions. Many people resort to artificial feeding because they believe
the conditions are unfavorable, and even when the mother's milk is given,
it is so seldom in accordance with natural conditions, that even these
infants do not develop normally. It should be our task to educate people
to believe that mother's milk is the proper food for the child, and that it
alone affords a positive guarantee for the normal development of the
infant. At the same time it should also be our aim to carry out researches
in the hope of discovering more satisfactory substitutes for the mother's
milk when it is not available. As we have said before, the nourishment
of the child should in no case be regulated solely with regard to the fuel
value of the food. The most important thing is to make sure that it will
serve for the construction of tissue. A milk substitute may be absolutely
worthless in spite of the fact that it has a high calorific value. Especially
at this time of rapid development the Law of the Minimum holds in its
entirety. By no means should the nature of the organic constituents
alone come into consideration. It is equally important that the inorganic
requirements should be satisfied. Furthermore, it is not even sufficient
to know the total amount of the inorganic material.

In the case of mammals the milk nourishment continues only during
the lactation period, and is then abandoned entirely within a relatively
short time. We have already seen in studying the iron content ' that

Cf. Lecture XVII, p. 386.

GENERAL METABOLISM.

659

it would be dangerous to continue milk as the sole food for too long a
time. In the case of the human race, milk plays a more or less important
function as food during the whole period of growth and even for adults.
The utilization of the material in milk is somewhat greater on the part
of the infant than is the case with the adult, but even then it remains
very satisfactory. Cow's milk is utilized to good advantage by the human
offspring.

One very important food for the growing organism, as well as for the
adult, is the egg. In the raw state, as well as when hard boiled, the egg is
equally well utilized. As far as we are concerned, the eggs of birds alone
come into consideration, and especially those of hens.

In 100 grams of fresh eggs there are present : 73.7 grams water, 12.6
grams nitrogenous matter, 12.0 grams fat, 0.7 gram nitrogen-free extrac-
tive substances, and 1.07 grams of ash. The percentage composition of
the last-mentioned is as follows : '

Per cent

Ash of
the Dry

K2O

NazO

CaO

MgO

FezOa

P2O5

SO3

SiOj

CI

Material.

Total contents

of hen's eggs

3.48

17.37

22.87

10.91

1.14

0.39

37.62

0.32

0.31

8.98

White of the

eggs . . .

4.61

31.41

31.57

2.78

2.79

0.57

4.41

2.12

1.06

23.32

Yolk of the

eggs . . .

2.91

9.29

5.87

13.04

2.13

1.65

65.46

0.86

1.95

The greater part of the phosphorus contained in eggs (about 80 per cent)
is present in lecithin, nuclein, and other organic compounds. Only a
small part, is found in the form of inorganic salts.

Flesh foods form one of the most important articles of diet for adults.
The amount eaten varies with different nations and with different classes
of people. According to Ostertag^ the consumption per head in different
localities is as follows:

Per year in kgins
Per day in gms.

Australia.

U.S.A.

Great
Britain.

France.

Belgium

and
Holland.

Austria-
Hungary.

Russia.

Spain.

111.6
306

64.4
149

47.6
130

33.6
92

31.3

86

29.0
79

21.8
59

22.2
61

10.4
29

In China even less meat is eaten than in Italy. Similarly certain negro
races live almost entirely upon vegetables. The composition of meat as

' Konig: loc. cit. p. 576.

' R. Ostertag: Handbuch der Fleischbeschau, p. 4. Stuttgart, 1899.

660

LECTURE XXVIII.

it comes upon the table varies greatly. The species of animal fiom which
it is obtained, the amount of waste (tendons, bones, etc.), and the state of
nourishment of the animal, all constitute important factors. Meat con-
tains, besides albumin, other nitrogenous substances, such as creatin,
creatinin, sarkosin, xanthin, and carnin. It is not right, therefore, to
estimate the amount of protein present by the nitrogen content alone.
The composition of the flesh of fish is quite similar to that of mammals.
Fish is, as a rule, utilized by the human organism as well as other flesh
foods.

The values given in the following table illustrate the composition of dif-
ferent kinds of food:

Meat of mammals

Salmon

Pike

Shellfish ....

Sole

Oysters

Lobsters ....
Fresh-river crabs
Edible snails . .

Nitroge-

Water.

nous
Material.

Fat.

76.0

21.5

1.5

64.0

21.1

13.5

79.6

18.4

0.5

81.5

16.9

0.3

82.7

14.6

0.5

80.5

9.0

2.0

81.8

14.5

1.8

81.2

16.0

0.5

80.5

16.3

1.4

1.0
1.2
1.0
1.3
1.4
2.0
1.7
1.3
1.3

Meat as well as milk is used in the manufacture of certain prepared
foods. The latter is used to make butter and cheese; the former in the
manufacture of sausages and other cured products. It may be said that
the value of flesh food from an economic standpoint is largely dependent
upon the amount of the product which may be utilized. With fish, for
example, there is a relatively large amount of waste.

Among the vegetable foods the different kinds of grain are very impor-
tant; these are used in the form of meal and flour in a number of different
ways, especially in bread-making. They also serve, as well as potatoes,
for the manufacture of starch. Then again there are the large number of
green vegetables and fruits. As regards their nutritive value and utiliza-
tion in the human organism, we have found that on the whole they are not
utilized as completely as the flesh foods. A vegetable diet gives rise to a
large amount of e.xcreta. In the case of the different grains it makes con-
siderable difference whether the whole grain is used in the flour, or only
that which has been freed from the hulls. The more of the hull there is
present, the le.ss the percentage utilization. We cannot include within
the scope of these lectures all the different data which have been acquired
concerning these relations, and which are so important in considering the
food-supply of a people. We shall have to refer to the special works on
the subject. Here we only desire to point out the great importance of the

GENERAL METABOLISM.

661

study of such problems for the complete understanding of the principles of
practical nutrition.

It is very significant that even the adult organism requires nutriment
not only as fuel, but also for the constant construction and renewal of the
cells. It must never be forgotten that the organism is adjusted for a
mixed diet, and that certain kinds of stimulation are necessary for the
production of the digestive juices. It is not at all practical to consider
replacing our food with chemical products. Our knowledge regarding
the necessary nourishment for the organism is far too limited for us to
attack such problems. The attempt has been made quite recently to
provide albumin in as pure a form as possible for the use of the sick. Our
understanding of metabolism is not such that we can approve of such
experiments unreservedly. In paying a good deal of attention to a par-
ticular nutriment we are pretty sure to neglect something else. We have
seen from the Law of Isodynamics that the carbohydrates and fats replace
one another in accordance with their calorific values, and that protein may-
be replaced by these two foodstuffs to a certain extent. There is always
danger, in making use of chemically -pure foods, that too little attention
will be paid to the amount of inorganic salts' which are required. The
knowledge of the calorific requirements for the performance of a definite
amount of work is very important for the establishment of a ration. The
calorific requirement forms a foundation. It must not, however, be re-
garded as the sole requirement. The composition of the ration is by no
means a matter of absolute incUfference.

In the following table the calorific values of a few foodstuffs are given: '
one gram of substance yields the following number of heat units, expressed
in small calories :

(A) PROTEINS.

Stohmann.

Berthelot.

Stohmann.

Berthelot.

Plant-fibrin ....

5941.6

5832.3

Flesh fibers (with fat

removed)

5720.5

5728.4

Serum-albumin . .

5917.8

Flesh (with fat rera'd)

5662.6

Hemoglobin ....

5885.1

5910.0

Blood-fibrin ....

5637.1

5529.1

Milk casein ....

5867.0

5626.4

Peptone from blood-
fibrin

5298.8

Yolk of egRs . . .

.5840.9

Chondrin

5130.6

5342.4

Legumins

5793.1

Vitellin

5745.1

5780.6

Egg-albumin . . .

5735.2

5687.4

(B)

F.\TS.

Tissue fat

9484.5

Linseed oil

9623.0

Butter

9231.3

9328.0

Cf. Koiiig: loc. cit. p. 2S3 et seq.

662

LECTURE XXVIII.

(C) CARBOHYDRATES.

Grape-sugar
Fruit-sugar
Galactose .
Cane-sugar .
Milk-sugar .

3742

6

3755

0

3721

5

3955

2

3951

5

Maltose .
Starch .
Dextrin
Cellulose

3949.3

4182.8
4112.5
4185.4

(D) ORGANIC ACIDS.

Oxalic acid. ,
Tartaric acid

571
1745

Citric acid
Benzoic acid

2397
6281

This is all that we care to mention with regard to metabolism as a whole.
We realize that we have merely touched upon most of the questions
without attempting to consider them from all the different points of view.
Metabolism physiology has during recent years developed a field of its
own. In pathology it finds much that is kindred in nature. Both fields
are intimately connected with one another, and this is largely the result
of recent efforts. Without going into clinical experience in detail, it would
be hardly possible to give a complete picture of general metabolism
from all directions. We shall, therefore, be obliged to refer the reader to
the special works on the pathology and physiology of metabolism. Our
discussion has been only to bring out the more important principles, and
will, it is to be hoped, prove an incentive to further studies.

LECTURE XXIX.
OUTLOOK.

We have not even apprqximately exhausted the large domain of
physiological-chemical investigation in the discussion of our knowledge
concerning the chemical processes which take place in plant and animal
organisms. We have merely been able to touch upon the fundamental
principles upon which the science rests. To be sure, our knowledge is still
incomplete, and the explanation of many phenomena has resulted solely
from the play of the imagination. On the other hand, the progress of
the exact sciences is constantly bringing new methods to the aid of physi-
ological chemistry, and in this way we are being led to more definite prob-
lems, so that we are hopefully looking forward to the further development
of the field. Little by little the unknown becomes the known. Direct
proofs gradually replace the indirect conclusions. The physiological
chemist is gradually breaking away from the observation of a single
individual. It is becorhing very evident that satisfactory results can
be obtained only when the investigation is carried out with as many dif-
ferent organisms as possible. The broader the foundations, the greater
the scope of observation, and the more varied the conchtions are under
which certain physiological processes are studied, the less danger there
is in arriving at biased conclusions. Just as morphology developed into
an independent science only by extensive comparative investigations
and by the careful consideration of the anthropogeny of each individ-
ual species, so we shall expect to obtain from comparative physio-
logical-chemical investigation the answer to many problems and to
receive new impulses for further inquiry. Just as an organ which is
functionally unimportant — e.g., an apparently superfluous bone — may
be in the eyes of a zoologist an eloquent proof of a common origin with
a certain class of animals, and just as the botanist is able to conclude
from the similarity of the flora of our highest Alpine peaks and that of
the Far North that there is an intimate relation between these two
regions, so we may certainly hope to meet here and there with chemical
processes which will lead us from the present into the far-distant past.
What an infinite perspective is opened to us by a ghmpse at the wonderful
flower tapestry of the Alpine heights, this so foreign and so characteristic

663

664 LECTURE XXIX.

witness of long- forgot ten ages! We also find many insects which are sharply
confined to the Alps and to the Far North. Many a paleontologic discovery
serves to form a bridge between two apparently foreign fields, and at one
stroke changes assumptions to indisputable proofs. The bottoms of our
Alpine lakes are covered with forms of life which we encounter again only
in the arctic regions.' How interesting it would be to turn our physiologi-
cal chemistry into similar channels! We have hardly begun to advance
in this direction, for our present methods are still unable to follow the flight
of thought, and our understanding of the chemical processes taking place
in the different organisms Ls still too limited for us to make comparative
studies. Nevertheless this goal should be regarded as most worthy of
attaining. To be sure, there are a great many isolated facts and a multi-
tude of observations concerning the organisms of different kinds of plants
and animals, but they are far from being of equal value, and it remains to
unite our knowledge of certain processes into a continuous chain. We
can, however, call attention to certain facts which indicate that not only
every species of animal but even each individual is to be regarded as one
which is characteristic and limited in its general metabolism.-

Let us consider for the moment the great multiplicity of forms in the
animal kingdom. In what a contrast the tissues of these morphologically
so different beings must stand! Consider the vertebrates. Everywhere
we find the same physiological function, the same tissue, the same organ.
Not only is this true of the external appearance, but even the finer struc-
ture shows a great similarity. In spite of this fact, the same organs of
different species of animals are very different in their metabolism, and
again the chemical composition of these organs and tissues must be char-
acteristic for each species of animals and perhaps for every individual.
Herein lies the reason for the differences in their metabolism. Let us see
what right we have to compare the purely morphological differentiation
of the various kinds of life into classes, families, and species with
physiological-chemical limitations, especially as regards the species. If
we consider physiological-chemical investigation as a whole, we shall find
that it extends in two directions. On the one hand many apparently
dissimilar elements are united by a common band to form a large whole,
and on the other hand many functions which were apparently identical
have, after careful study of the individual processes, been found to be
different in nature, and thus limitations have been found to e.xist where
none were suspected. Thus it was formerly believed that there was a

' Cf. F. Zschokke: Die Tierwelt der Schweiz in ihren Beziehung zur Eiszeit, Basel,
1901.

' Cf. Huppert: Ueber die Erhaltung der Arteigenschaften, Prag, 1896. Franz Ham-
burger; Arteigenheit und Assimilation, Leipsic and Vienna, 1903. Emil Abderhalden:
Naturwissenschaftliche Rundschau, 19, No. 44 (1904).

OUTLOOK. 665

great gulf between tlie animal and vegetable worlds. There was not
supposed to be anything in common between them as regards their
chemical processes and their metaboUsm. The plant cells were alone
assumed to build up organic substances, or, in other words, effect syntheses,
whereas the animal cells were assumed to break down only. Wohler's
discovery that benzoic acid is changed to hippuric acid in the animal
organism made the first breach in the wall separating the two kingdoms.
Then in rapid succession bridge after bridge has been built between these
apparently so distinct fields, so that to-day a common band unites the
animal and vegetable worlds. With this knowledge as a basis, it was
then desirable to study more closely the differences between these two
kingdoms, and also to unite them more closely by numerous intermediate
stages.

A few examples may illustrate the significance which, from a physio-
logical-chemical standpoint, governs the conception of the species.

The characteristic mark of distinction of mammals, the mammary
glands, deliver a secretion, the milk, which is quite uniform in nature.
The milk from an animal almost always has a very similar qualitative
composition, although it varies quantitatively somewhat. Each species,
however, has its own characteristic milk, and this is true not only of the
mineral constituents, but of the organic matter contained in it as well.'
In fact, we have reason to believe that even quaUtatively the different kinds
of milk differ from one another. Although our present knowledge does
not suffice to characterize these differences more precisely, — and indeed
the different kinds of casein appear to us as identical, — we must not forget
that we are never justified in deciding from the quahtative and quantita-
tive composition of the cleavage-products whether the original proteins
under investigation are identical. In the arrangement of these constit-
uents in the original molecule, to say nothing of the other kinds of isomer-
ism, there are countless possibilities.

Furthermore, let us consider the blood of different animals. In every
case it has the same function, the same physiological significance, and mor-
phologically the most far-reaching similarity. We always find blood-
corpuscles and plasma. What a remarkable similarity there is between
human blood and sheep's blood, and yet the sad experiences which have
resulted from the attempts at substituting the latter for the former have
proved that deep-seated differences must exist. The blood-corpuscles of
mammals all contain hemoglobin as a characteristic constituent. Its
function is invariably the same, and yet the hemoglobin is specific for
each different species, as is apparent from the external relations alone,
such as the crystalline form and solubility. Thus the hemoglobin of the

' Emil Abderhalden: Z. physiol. Chcm, 26, 4S7 (1899); 27, 408, 594 (1S99). Cf.
Lecture XVII, p. 404.

666 LECTURE XXIX.

squirrel crystallizes in the hexagonal system, and that of the mouse in the
orthorhombic. From a mixture of these two kinds of blood, each crys-
tallizes in its own specific form, and to the same extent as corresponds to
the original mixture.

Comparative quantitative analyses of different kinds of blood ' indicate
that, within fairly narrow limits, there is a definite composition of the
blood for every species. In closely related animals the relative amounts
of the individual constituents are similar, while in unrelated ones the difi'er-
ences may be very marked. It is noteworthy that the serum appears to
be of very similar composition in the case of all mammals. The product
in this case is apparentlj' identical when prepared from various classes of
different animals. We must not forget, however, that the examination
of the ash can give us at best only a rough idea of the composition of the
serum. It only serves to tell us what elements are present, and nothing
at all concerning the manner in which they are contained in the circulating
blood. But even if it were possible to prove that the inorganic and simple
organic constituents were qualitatively and quantitatively the same in
the sera of widely different species, there still remains the far more difficult
problem of determining the identity of the proteins. It is perfectly possible
that the different proteins in the blood contain groupings which are char-
acteristic of each species of animals.

Let us return to the oft-discussed observations concerning digestion.
We have seen that the nature of the proteins contained in serum ' is evi-
dently independent of the kind of food that is eaten. This is probably
true for all the other substances, or at least for the more complicated
organic ones. The cells of the body never know what the nature is of the
food eaten. They always receive a modified nutriment. The ferments
of the intestine and the accessory glands have the function of resolving
the complicated organic constituents of the food into cleavage-products
and, on the other hand, the cells of the intestine have the property of effect-
ing a chemical reorganization of these building-stones into new products
which are suitable for the cells of the body. The intestine thus regulates to
a certain extent the general metabolism and guarantees the maintenance
of a constant composition of our tissues. It is, therefore, perfectly clear
that the entire course of the metabolism in the cells of our organs is depend-
ent upon their composition. The composition of the protein molecule,
or perhaps better the protein molecules, is particularly influential in impart-
ing to each indi\ddual cell its characteristics. The cells of the body pro-
duce the ferments, and these are probably transformation products of
the proteins. We can easily understand that their finer construction is

' Emil Abderhalden: Z, physiol. Chem. 23, 521 (1897); 25, 65 (1898).
= Emil Abderhalden and Franz Samuely: Z. physiol. Chem. 46, 193 (1905). Cf.
Lecture X, p. 212.

OUTLOOK. 667

dependent upon that of the cell proteins. Thus their activity is regulated
very delicately, and the foundation is laid for a specific cell-metabolism.
The original structure of the cell determines its characterization for the
whole of its existence. Although we do not doubt that the various tissues
possess, corresponding to their functions, variously constituted cells, the
differences of which are apparent not only in the metabolic end-products,
but especially in the nature of the secreted substances, we can, on the
other hand, imagine that all the cells of one and the same nature have com-
mon outlines along certain hues, so that a common character marks the
whole cell structure of an individual.

Now an individual results from the union of two cells, the egg and sperm-
cells of the same nature. Each must contain within itself the common
form of cell composition, and thereby possess the kind of cell-metabolism
which is characteristic of this particular species. All the cells which result
in rapid succession from the fertihzed egg-cell can only assume these char-
acteristic tendencies, and thus the chemical unity of the original cell guar-
antees the maintenance of the species. This is subsequently maintained
by the activity of the intestine, which only permits such material to reach
the tissues as has been previously prepared in a definite manner for the
entrance into cell-metabolism and into the cells themselves. We do not
mean to assert that the body cells have lost the ability themselves to trans-
form and adjust to their composition and to their metabolism any foreign
nutriment, or especially any foreign protein. The unicellular organisms
must be able to cause all these processes to take place side by side. With
the higher animals, the function of transforming the foodstuffs is relegated
almost exclusively to the intestine. There is here a division of labor.*
We can easily imagine that by reason of an imperfect function of the intes-
tine, an insufficient amount of prepared material may be carried to the
tissues, and that under some circumstances the entire chemism of the
cells, their structure and at the same time their metabolism, may become
altered so that finally degenerations result which are apparently
inexplicable.

In mammals the individuality of the cells of the species is in a very great
measure guaranteed by the longer or shorter period during which the new
being remains associated with the organism of the mother. The foetus
obtains its nourishment from the blood of the maternal organism; the
suckling from her milk.

We have thus arrived at a purely chemical explanation for the concep-
tion of species and its maintenance. We admit that we are reasoning
from a limited number of observations, and are not yet in a position to
demonstrate experimentally the truth of our conception. We realize fully

Cf. Ulrich Friederaann and S. Isaac: Z. exper. Path. u. Therapie, 1, 513 (1904).

668 LECTURE XXIX.

that we are now only constructing as it were a scaffolding which will per-
haps serve to lead future investigation into definite channels.

We are not alone in this idea. Franz Hamburger/ starting from entirely
different experimental results, has likewise traced in a most interesting
manner "the individuality of a species and its maintenance to a definite
composition of the cells and body fluids. This idea is closely related to the
so-called biological reaction, which we shall discuss briefly. Its discovery
is associated with the names of Bordet, Tchistowitsch, and Nolf.^ It rep-
resents merely a generalization of the Law of Immunity, and depends upon
the formation of very specific substances after the introduction of products
foreign to the species. The knowledge of this principle is of great signifi-
cance for the further development of physiological chemistry. It forms a
bridge to the domain of pathology; and we become more and more con-
vinced that pathological processes are not sharply distinct from physio-
logical ones, but are common manifestations of body cells under definite
conditions. The limitations of purely physiological-chemical investiga-
tion are thus being more and more eliminated. By the improvement of
methods, it continually enters new fields, and on the other hand other
fields constantly attach themselves to it, and await new impulses for
further fruitful work. Here we must introduce the name of an investigator
to whom, more than any one else, our thanks are due for the expression
of this unity between physiological and pathological processes, namely,
Paul Ehrlich. We shall return to his theory, which has served as a founda-
tion for important investigations in this domain. In this connection also
we must call attention to the great importance of Pawlow's work.' He
likewise clearly recognized the numerous transition stages between physio-
logical and pathological processes, from his observations on the functions
of the alimentary tract under varying conditions.

It is quite out of the question for us to give here even a brief summary
of all the investigations which are based upon the conception of the " bio-
logical reaction." In a very short time it has developed into an important,
independent branch of biological science. We shall here very briefly call
attention to a few fundamental experiments. If we inject, for example,
horse-blood into a rabbit, the serum of this animal soon shows characteristic
new properties towards the injected blood. It dissolves the blood-corpuscles
and forms a precipitate with the new serum, called the precipitin-forination.
This reaction is a specific one. The serum of the rabbit which has been

' Loc. cil.

2 Jules Bordet: Annales de I'lnstitute Pasteur, 1899, 240. Tchi.stowitsch: Ibid.
1899, 41.3. Nolf: Ibid. 1900, 299. Cf. Rostoski: Zur Kenntnis der Prazipitine. Wurz-
burg, 1902.

■" Pawlow-Walther: Das Experiment als zeitgemasse und einheitliche Methode medi-
zinischer Forschung. Bergmann, Wiesbaden, 1900.

OUTLOOK. 669

treated previously with horse's blood will not react, for example, upon
the blood of the ox, sheep, or goat. We may add that the formation of such
specific products is not peculiar to the blood and its serum. The property
is common to all the cells, body-fluids, and secretions. If we inject the
spermatozoa of the sheep into a rabbit, the blood-serum of this animal
when added to the living, motile spermatozoa of the sheep will restrict their
activity. And this serum also has a solvent action upon the blood-corpus-
cles of the sheep's blood; i.e., the effect of the spermatozoa is the same as
if sheep-blood itself had been injected. Hamburger assumes that every
cell, and all the other substances which circulate in the body-fluids, possess
definite atomic groupings, which we must regard as imparting the specific
nature to these products. Consider the ferments! These are substances
of which we know merely the effect. Emil Fischer has, as we have often
mentioned, called our attention to their very specific action, and indicated
clearly their dependence upon the configuration of the different compounds
with which they react. The ferment molecule must posse.ss certain definite
groups by reason of which it can react with certain other molecules,
and only these. Fischer has aptly compared the relation between the
ferment and the compound with which it reacts as that of a key, and the
lock which it fits. Just as a given key fits only a certain kind of lock, and
conversely the lock can only be opened by just such a key, so the specific
atomic grouping of the ferment molecule probably harmonizes exactly
with the compound to be acted upon. We can easily imagine that slight
changes in the arrangement of the atoms in the ferment molecule will be
sufficient to modify the efficiency of the ferment. On the other hand, we
can also believe that when this characteristic gi'oup is in combination with
some other substance, the ferment will be prevented from exercising its
function. It is possible that the zymogen stage of the ferment is brought
about by some such combination or different arrangement of the atoms
in the ferment molecule. These views are advanced merely to indicate
that just as the individual cells can produce ferments, they them-
selves may be endowed with specific atomic groups, which, as Ehrlich
has suggested, may perhaps stand in certain relation to the assimilation
of the food. In this case there would be a certain analogy between fer-
mentation and cell-activity. We know of the formation of anti-ferments
when the ferments are introduced into the circulation. These anti-
ferments are also specific in their action. We may look upon the forma-
tion of the precipitins, and related bodies, as taking place in a perfectly
analogous manner. We introduce into the blood and cells of a foreign
species of animals a certain atomic grouping which is peculiar to a different
species, and which is perfectly foreign to the first animal. The animal
organism reacts towards this exactly in the same way as towards the
toxines which result from micro-organisms. Sul)stances are evidently

670 LECTURE XXIX.

produced which are so constituted that they exactly correspond to the
structure of the foreign product. In this way the active groups which
would be injurious to the body-cells are rendered harmless. The "bio-
logical reaction" of the animal organism is, from this point of view, to
be regarded merely as a means of protection.

Let us see what subsequent investigation has done for us in this direc-
tion towards establishing our conception of species. In the first place
it could be shown that the formation of precipitins was not confined to
one species, but that the specific result of the reaction was limited to
related animals within such narrow limitations that the relationship
between animals which from morphological and other similarities are
usually grouped together could be confirmed by means of the "biological
reaction. " Nuttal ' found, for example, that the serum of a rabbit into
which the serum from dog's blood had been injected would give a pre-
cipitate with the blood of eight difi^erent kinds of Canidce, but not with
the blood of any other species. Friedenthal ^ showed, furthermore, that
only the anthropoid apes showed a marked blood-relation to man,
whereas the lower apes showed but slight indication of a common
origin. -We may add that the different kinds of birds have also been
compared in this way, and that recently Uhlenhut ' has succeeded in so
perfecting the method of carrying out the biological reaction that it has
become possible to differentiate and distinguish between closely related
kinds of blood. The reaction has become of considerable importance in
forensic blood determinations.

It would be, of course, desirable to ascertain what compounds the cells
and body-fluids make use of for carrying out this specific reaction. We
would naturally think of the proteins in this connection, for, on account of
their extremely complicated composition, they are most suited to serve as
carriers of specific groups of atoms. As a matter of fact, it has been found
possible to prove that after the injection of protein, e.g., serum-albumin,
perfectly specific precipitins are formed;* and, indeed, it was even found
possible to distinguish by this means between the casein of different kinds
of milk. We do not yet know whether in such cases the individual protein
substances come into consideration, or whether the effect is produced by
impurities which adhere to them. At all events, it is of great interest to

■ Cf. Nuttal: Proc. Roy. Soc. 69, 150 (1901). Blood Immunity and Blood Rela-
tionship. Clay & Sons, London, 1901.

' Arch. Anat. Physiol. 1900, 494. Sitzsber. Berliner Akad. 1902. Verhandl. Ber-
liner physiol. Gesell. 1904. Uhlenhut: Arch. Rassen- und Gesellschaftsbiologie, 1, 682
(1904).

' Uhlenhut: Deut. med. Wochschr. 1905, 42.

* Cf. among others, L. Michaelis: Deut. med. Wochschr. 1902, 41. L. Michaelis and
Carl Oppenheimer: Arch. Anat. Physiol. Sup. 1902, 336. F. Obermayer and E. P. Pick:
Wien. kliu. Wochschr. 1904, 10. Andrew Hunter: J. Physiol. 32, 327 (1905).

OUTLOOK. 671

find that if the proteins enter metabohsm in any other way than through
the intestines, i.e., in such a way that the sulDstances have not lost their
identity by the activity of the intestine and changed so that they are
suited to the body-albumin, then the only effect to be observed is the for-
mation of these specific products. This result supports our views regarding
the great importance of the digestion processes and assimilation in the
intestine for the maintenance of the species.

With the assumption of atomic groups of specific nature in the individual
cells, and thus in the egg and sperm, new aspects are given to the problem
of heredity. Although it has not yet been found possible to cause mor-
phological changes that have been artificially produced to be inherited,
still there remains the possibility of effecting hereditary variations by
influencing the chemical composition. We may mention the interesting
experiments of Engelmann and Gaidukow ' who succeeded for the first
time in proving satisfactorily that a property which had been acquired
could be inherited. If cultures of Oscillaria sancta, a kind of alga, are kept
for months at a time in light of a definite color, then the single threads of the
alga gradually assume a complementary color, i.e., a shade which is favor-
able to the assimilation in such light. The change of color takes place only
with the living organism. Aqueous solutions of the dye do not show any
such change in shade under the same conditions. We have, therefore, a
case of a vit,al, physiological adjustment. Engelmann designates it as
chromatic adaptation. Now, strange to state, this acquired change of color
is retained when the Oscillaria are placed in ordinary light. In the case
of rapid propagation, the new color prevails, so that the assumption may
be made safely that there is a new formation of chromophyll in the younger
generations of cells. In reality we have here a ease of the inheritance
of a change in chemical composition, and in fact in the formation of a
pigment, the sj^nthesis of which remains the same as formerly in the new
environment.

It might have been thought that by feeding compounds of quite definite
composition it would be possible to effect a change in the chemical compo-
sition, and thereby in the cell-metabolism. Such experiments must be
without much prospect of success, because of the fact that, as we have
seen, the intestinal wall frustrates the entrance of such foreign substances.
The unicellular beings are likewise unsuitable for deciding such questions,
because they are also provided with the necessary means for maintaining
their constancy of chemical composition. At best, the only way we can
conceive of any such experiment being successful would be to continue for
a long time the introduction of such substances to the body-cells in some
other way than through the ahmentary canal, for it would be expected that

' T. W. Engelmann: Arch. Anat. Physiol. 1902, Suppl. 333. Sitzsber. Berliner Akad.
Wissenseh. 1902. Arch. Anat. Physiol. 1903, 214.

672 LECTURE XXIX.

in the course of time tlie cells would lose to some extent the ability of trans-
forming rapidly the substances which are brought to them. At all events,
this is the only way in which the metabolism of more highly organized
beings could be affected.

It is almost generally assumed that in the inheritance of certain proper-
ties the nucleus of the cell plays a particularly important part, and in fact
it is usually assumed that it alone is able to transmit the characteristics of
the parents. This is certainly not justifiable, for although the protoplasm
represents an apparently homogeneous mass, wliich is but slightly differ-
entiated and excites but little the interest of the histologist, it is not at all
apparent why, with its infinitely complicated composition, it should not be
capable of at least taking part in the processes named. In this direction the
following experiment of Godlewski ' is interesting. He fertilized nucleus-
free fragments of Echinus eggs with spermatozoa of Antedon rosacea. It
was found possible in some cases to effect a development. Even these
nucleus-free pieces did not develop to be of the Antedon type. In order
to understand thi.s experiment better, we should recall the investigations
of Loeb,^ which we have previously mentioned, who succeeded in a great
number of cases in bringing eggs to spontaneous segmentation by the
action of certain salts in definite concentrations. Of great interest is his
discovery that under certain definite conditions the egg of a definite species,
e.g., that of a star-fish, could be fertilized by the spermatozoa of an entirely
different nature. We shall not go into the significance of these discoveries
any more deeply, but will briefly consider the outlook which Loeb himself
obtained from his experiments. He believes it is not at all improbable
that the greater part of the many different forms of life, particularly those
of deep sea, may have resulted from conditions which are not at all unhke
those of his experiments. It is indeed conceivable that in the course of
time the composition of the sea-water in certain localities may change so
that the conditions for the fertihzation of one species are replaced by those
corresponding to a different one. We mention these experiments merely
to illustrate in what widely different ways biology seeks to penetrate the
mystery of life. To be sure, we are not justified in considering the inter-
esting results of Loeb's experiments as by any means solving the problem of
egg development, or even in hoping that by such a way we shall obtain an
insight into the laws of heredity. Loeb's experiments merely show that it
is possible, by changing the concentration of a salt solution in which the
unfertilized egg is placed, to bring about cell-division. They do not tell
us anything at all about the causes of cell-multiplication, and the reason

1 Anzeigen der Akad. Wissensch. Krakau, 1905, 501.

2 University of California Publications, 1, No. 1, p. 1 (1903) ; 1, 39 (1903) ; 1, S3 (1904) ;
2, 5(1904). Pfluger's Arch. 99, 323 (1903); ibid. 104, 325 (1904). Cf. Emil Abder-
halden: Arch. Rassen- und Gesellschaftsbiologie, 1, 656 (1904).

OUTLOOK. 673

for the regular arrangement and the gradual differentiation of their
functions.

Similarly, Godlewski's investigation fails to shed light upon the
problem of heredity itself. It shows us, on the other hand, quite clearly
and definitely that it is not right to regard the process of cell-division as a
function of the nucleus alone, and consider that the protoplasm plays a
passive part. The cell of the egg, in its entirety and according to its chem-
ical composition, must have, more than any other cells of the body, the
ability to subdivide and increase. The entire nature of cell propagation,
in all its particulars, has been found to be bound up with it.

Loeb's experiments have shown that the egg-cells of various species of
animals may be easily induced to take^jart in such a process of cell-division.
The cells of one kind develop up to a certain stage apparently of their own
accord, othei-s require a slight stimulation, while others need quite a con-
siderable impulse. The entire development of the individual is bound up
with countless problems. When we remember that we have before us a
mode of development which has been inherited for ages and has been
taking place over and over again in paths which have been well defined,
the wonderful thing is that there is apparently no limitation to it. We
can understand perhaps how, in the case of the mature organism, lost
tissues may be regenerated; and, again, it does not appear so remarkable
to us that from the cells of a certain tissue others may be produced with
the same function and the same chemical composition, so that the whole
corresponds to a morphological and functional unit. On the basis of our
present knowledge, however, it is a mystery how all the different tissues
can develop from a single cell, and how in each kind of tissue chemical
processes will take place which are peculiar to that particular tissue, as is
evident from a study of the secretions and of the end-products of their
metabolism. The complicated picture of the morphological development
of the individual seems to us far less remarkable than the much more-
intricate and more involved differentiation of the chemical construction
and the chemical processes which take place in the individual cells.

Now we know that the individual, in the case of the more highly organ-
ized animals especially, shows not only a development of species, but in
its beginnings there are stages of development to be detected which we
can understand only from the history of the ancestry of the animal organ-
ism. We need recall merely the formation of the gill-clefts or gill-slits, a
stage of development which even the human foetus passes through. Is it
not probable that there are differences in the chemical composition of the
tissues during the separate stages of its being, which are likewise suited
for tracing the entire group of vertebrates back to a common ground
plan? We must thank G. von Bunge for an answer to this question.*

Z. physiol. Chem. 28, 4.52 (1S99).

674 LECTURE XXIX.

He pointed out that the land vertebrates are richer in sodium chloride
in proportion as the stage of development is young. This fact was illus-
trated particularly clearly by comparing the sodium chloride content of
the cartilage of embryo and that of later stages of development of the
same species. The older the animal, the lower sinks the content of
sodium and chlorine. There must be some reason why the cartilage of
the embryo is so rich in common salt. This fact is all the more striking
because the land is poor in this salt, potassium salts preponder-
ating. Typical inhabitants of the land, such as insects, have in
their bodies the elements sodium and potassium in about the same
proportion as that in their food. The remarkably high content of sodium
chloride in the cartilage of the early stages of development of verte-
brates which inhabit the land may be regarded, like the appearance
of the gill-slits and other similar phenomena, as an ancestral reminis-
cence. It is not at all strange that even the chemical composition
should indicate long-forgotten conditions.

Not alone the development of the individual leads to such various
problems, but in later life also we meet with processes the nature of which
we can understand only very imperfectly. We usually assume that the
development of the entire organism can be traced back eventually to three
germ-layers. We can indeed distinguish these as regards their chemical
function and their construction even although one and the same layer,
as the ectoderm, may be of very heterogeneous construction. We can
imagine that the cells of each of these three germ-layers among themselves
have certain- common characters so that eventually each individual cell
is in a position of replacing other cells which have resulted from the
same germ-layer and even to form these anew. It is not sufficient, how-
ever, to conceive that these three germ-layers correspond to three great
classes of different cells. They must, as we have already suggested,
all show common characteristics. To what extent the body-cells of
even adult organisms have the ability of changing their composition,
and thereby their function, is shown by the following experiment.' If
the crystalline lens is entirely extirpated from a water salamander, after
a short time there will be found in its place a newly formed transparent
structure which perfectly corresponds to the original lens. It is very
interesting to find that the new formation of the lens starts from the
epithelial cells of the iris. Now the cells of the latter are nornially as
opaque as possible. Nevertheless these cells multiply after the removal
of the lens, and the pigment, which causes the opacity, disappears. In
this process comprehensive chemical processes must take place. The cells

' Cf. Vincenzo L. Colucci: Meraor. della R. accad. delle scienze dell' inst. di Bologna,
Serie 5, T. 1, p. 593 (1890). Also G. WolfE: Arch. Entwicklungsmechanik der Organie-
men, 1, 380 (1896).

OUTLOOK. 675

of the crystalline lens and of the iris have unquestionably quite different
construction corresponding to their different functions. Certainly their
metabohsm is different.

Thus when we find that from one tissue another of entirely different
characteristics may be formed, with a quite different function, we can-
not help asking whether pathological new formations do not arise from
similar processes. Certainly the cells of the newly-formed crystalline lens
of the Triton renew themselves and form cells of the same kind, and show
hardly any tendency to form the pigment which is required by the cells in
the iris. Similarly it is possible that a body-cell may retain its individual
nature only with difficulty if for any reason its chemical nature and func-
tion become seriously altered, and the progeny of such a cell will possess
the characteristics of the mother-cell so that gradually a whole cell-complex
will develop which is of a nature foreign to the entire organism and to its
metabolism, and in fact the metaboUc end-products of this new cell-com-
plex may exert a disturbing influence upon the metabolism of the remain-
ing cells of the body.

Apparently there is a connection between these cells and the mother
soil — we refer especially to sarcoma and carcinoma — for it has been
frequently doubted whether such cells can be successfully transmitted to
organisms of a different species. We are, in making these suggestions,
very far from explaining the formation of these peculiar, atypical
tissues. We only wish to bring forth the fact that with the further
development of our physiological-chemical knowledge new tasks will be
set, and that even problems of purely morphological investigations will,
in the course of time, become closely allied to those of physiological
chemistry. If it is once found possible to compare the metabolism of the
cells of a cancer, or other mahgnant growth, with normal cells, we may
certainly expect to obtain a more accurate insight into the nature of such
mysterious processes.

Taking into consideration all that we know concerning metabolism,
and what we have deduced indirectly, it does not appear to us impossible
that, among the more complicated processes, here and there a link in the
chain of separate processes may be missing, or react in a faulty manner,
thus giving rise to degenerations which eventually may be inherited, or at
least there may be indications of the transmission to several members of a
family. We would recall certain anomalies,' such as cystinuria, alcap-
tonuria, and albinoism, and finally familiar types of degenerations in the
nervous system. Gout and diabetes may also be caused by disturbances
in some phase of the functions of cell-metabolism which are partly
inherited and partly acquired. Although we may imagine that, for
example, an anomaly in the composition of the body-albumin is inherited

' Cf. A. E. Garrod: Pfliiger's Arch. 97, 410 (190.3).

676 ■ LECTURE XXIX.

in such a way th'at the egg-cell, as a part of the mother's organism, is
affected by the disturbance, still this will not hold for the transmission of
many other properties. In this direction we have the peculiarity in the
inheritance of hemophilia, as we have already indicated.

If we consider the cells in their entire chemical construction, and con-
stantly bear in mind that herein lies their entire function, we can indeed
imagine that a faulty organization of the cell will have an influence upon
the general metabolism. The conception of the disposition which plays
such an important part in pathology is certainly well founded, and the
cause of it is to be sought in the state of the function of certain groups of
cells, or the whole cell state of the body may be afTected in such a way that
the individual appears to be functionally deficient. Naturally such spec-
ulations, which have no experimental justification, do not penetrate at all
into the nature of a disposition; but, nevertheless, it seems fitting to sug-
gest that an alteration in the chemical processes of the cells themselves
may come into consideration here.

Let us now turn back to the limitations of the concept of species on
a basis of physiological-chemical investigation. We have mentioned only
a few of the species which have been most thoroughly studied. We might
multiply these examples, but a few suggestions should prove sufficient.
We know that the general metabolism of different animals is unlike.
This is partly due to the different kinds of nourishment which they receive.
Thus we can readily imderstand that the urine of herbivora will be of
quite different composition from that of pure carnivora, and that of
the latter will likewise be different from that of omnivora. We even
find differences in the same animal species. We recall in particular the
amount of kynurenic acid present in the urine of dogs. We encounter
other characteristics which indicate that each species of animal has a
peculiar cell-metabolism. We do not doubt at all that there are also
individual differences. The fact that there is a perfectly definite endogenic
uric acid value for each individual points in this direction. Also the pecul-
iar characteristic color of the sldn, hair, eyes, etc., gives one the impression
that it is the expression of a speciaUzed individual metaboUsm of certain
definite cell-complexes. Each individual possesses its own characteristic
smell. This is usually not observed by "human beings, but that there are
such distinctions is evident from the abihty of certain animals, such as the
dog, to trace a person by the scent.

The differentiation of the species increases infinitely in the case of lower
animals, especially the anthropods. What a richness of coloring, and what
splendor in the groups of butterflies and beetles! Each separate color is an
expre.sf3ion of the chemism of the cells producing it. We meet with the
same phenomena in the plant world, where the beautifully colored flowers
indicate an unsuspected variety of peculiarly modified chemical processes.

OUTLOOK. 677

To be sure there are certain relations between the pigments that the vege-
table kingdom produces and those of the living animal organism, which
find expression particularly when it is a case of assuming a color for pur-
poses of protection. It would be interesting to compare the pigment used
in mimicry ■ftdth that of the plants upon which the animals feed. We
would also refer to the large number of poisonous substances which animals
produce partly as weapons of attack and partly for other purposes.

The specific character of cell-metabolism does not by any means stop at
causing differentiated incUviduals. It also concerns the individual cell.
We shall refer in this connection merel)^ to the specific metabolic sub-
stances, especially the toxines, which the cells produce to combat every
kind of bacteria.

The intricate, specific organization of the cells of certain kinds of animals
is also indicated indirectly, as, for example, by their behavior towards certain
poisons. Thus we know that a man who is not accustomed to morphia
will usually sleep after taking two centigrams, whereas with dogs, ten times
this amount does not have the slightest effect toward producing sleep. A
goat will stand twenty grams of morphine hydrochloride without showing
any desire to sleep, although other indications of poisoning appear.
Whereas atropine is a violent poison for man, rabbits are perfectly immune
toward it. They can feed unharmed upon leaves of belladonna. It is
also known that all kinds of animals do not furnish equally good fostering
soil for pathogenic bacteria, and that different organisms react differently
toward certain infections.

If we summarize all these details, we shall recognize how manifold the
processes are which stamp their outlines upon every kind of species and
every individual. Here lies before us an immeasurable, almost unculti-
vated field of investigation. New problems and new methods become
more and more intricate, and the demarcations of the conception of species
become more and more exact. The purely morphological boundaries of
species, families, and classes will pass away. Comparative physiological-
chemical investigation will, in the future, take the lead and control the
results of purely morphological investigation.

We must consider one other field which is gradually coming into more
intimate contact with physiological chemistry. We refer to pharmacology.
We are no longer satisfied with determining the effect of individual
drugs. We desire to determine, by comparative experiments, whether
it is this or that group which represents the active principle, and
whether the compound exerts its effect as such, or whether it first under-
goes a transformation.' Finally, we are interested to know how the

' Cf. S. Fraenkel: Die Arzneirnittelsynthese auf Grundlage der Beziehungen zwischen^
chemischen Aufbau und Wirkung, Berlin, J. Springer, 1906. H. Bechold and P. Ehrlich:
Z.physiol. Cham. 47, 173 (1906)^

678 LECTURE XXIX.

substance introduced is broken down, and in what form it leaves the body.
Conversely our interest is fastened upon the way in which the body reacts
toward the influence of certain compounils. Here also innumerable specific
cell reactions come into play, and we constantly meet with indications of
the functional differences of the cell-complexes of different organs. Investi-
gation in this field is only just beginning. We lack methods for tracing
the course of each individual substance in the body from cell to cell. We
should Hke to know whether the different body-cells have a different
affinity for certain products which are introduced, and whether perhaps
the specific reaction of the organism is not the expression of the ability for
selection on the part of certain particular tissue-cells. On the other hand,
we are interested, in every case, to know how the animal organism
wards off the action of all the different substances which are introduced
into the body and are foreign to it. In considering the functions of the
cells we constantly met with such problems and have seen in how many
different ways the organism protects its cells from the action of such sub-
stances. Sometimes the substance which is introduced is oxidized, some-
times it is reduced, and at other times it is conjugated either directly,
or after certain preparatory attacks, with different substances which
are produced in the intermediate metabolism. Thus we have seen how
the animal organism behaves toward glycocoll, sulphuric acid, urea, and
glucuronic acid.' We know that the proteins themselves play an impor-
tant part in these processes. They combine with many of the.se harmful
substances which are introduced into the body and form insoluble com-
pounds. Often this combination involves the breaking down of the
cell. The ceil-albumin becomes incapable of exerting its function. The
effect of a great many poisons is due to their affinity to the proteins of the
cells and tissues. Here again we meet with certain specific differences
according to the nature of the cell and the proteins which have taken part
in their construction. In this connection we would recall the different
coloring of the cells, the cause of which is likewise attributed to a different
chemical construction of the cells. It is this which lies at the root of
the various functions of the different kinds of cells, and the separate
organs, and upon it depend also all of the various reactions which have
been mentioned, whether the cell plays an active part, or whether a
mere passive one.

' Cf. E. Fromm: Die chemischen Schutzmittel des Tierkorpers bei Vergiftungen.
K. J. Trubner, Strassburg, 1903.

LECTURE XXX.

OUTLOOK.

II.

We have seen in discussing the boundaries of physiological-chemical
investigation, that such exist only artificially, and that the domain is
immensurable. The tendency is becoming more and more marked to refer
sciences which were formerly sharply defined to a common basis. This is
particularly true in the domain of pathology, which is becoming more and
more closely related to physiology. In fact, pathological processes in a
certain sense are nothing else than physiological processes of our body-
cells under specific conditions. This applies particularly to a group of
processes, interesting alike to pathologists and physiologists, namely, the
formation of certain substances in response to the action of other products
on the body-cells. We have already encountered these processes at various
times. We have seen that the animal organism responds to the intro-
duction of ferments by the formation of anti-ferments; that is, the cells
produce substances which prevent the activity of the ferments. We again
met this problem in considering the so-called " biological reaction."
Here also, it was a case of the formation of distinct metabolic products.
The most significant thing about these processes is the fact that the
products formed act in a very specific manner. We thus involuntarily
return to an analogous process which we have already mentioned. It is
known that the human and animal organism is capable, not only of
withstanding the infection of specific pathogenic bacteria, but also, that
it possesses peculiar characteristics, which prevent a second attack of the
same bacteria for a long time after their first repulse. This, of course,
only applies to certain infectious diseases. Other diseases do not leave
such a condition of affairs behind them. This is evident from the fact
that one may be afflicted several times with the same disease, — e.g.,
pneumonia, — whereas other sicknesses, such as typhoid or scarlet fever,
usually occur but once.

The far-reaching specificity is also noticeable in these cases. This is
best demonstrated by the following example. We can determine, by
experiment, the quantity of cholera bacteria necessary to cause the death
of a guinea pig of a given age and weight. If we take a smaller quantity,
the animal will only become sick, and will gradually recover. This guinea

679

680 LECTURE XXX.

pig has now obtained an entirely new property, which sharply differen-
tiates it from the other animals of the same class, which have not been
treated in the same manner. We usually speak of this as an acquired
immunity. This is evident from the following. The immunized animal can
then be infected with an amount of cholera bacteria which would formerly
have produced death, and it now does not even cause illness. If we
inject these bacteria into the abdominal cavity, and after a time withdraw
some of the fluid, we shall observe a very characteristic picture under the
microscope. The cholera bacteria have lost their activity, and form small
balls, which are sometimes collected into " clumps." A control animal, inoc-
ulated for the first time with these cholera bacteria, will show an entirely
different picture. The organisms in this case are endowed with great
activity. We find it necessary to assume that the immunized animal
possesses substances in its organism which injure the cholera bacteria,
and restrict their activity. This belief is strengthened by the investigations
of R. Pfeiffer.' If, together with the cholera bacteria, serum from an
immunized animal is injected into the abdominal cavity of another animal
which has not been previously treated, we shall observe the same phenom-
ena which were characteristic of an animal which had already been infected
with cholera bacteria. The latter become non-motile, and form balls or
clumps. M. Gruber^ has shown that this phenomenon may be verified
in a test-tube, by bringing cholera bacteria into contact with serum from
an animal infected with cholera. We immediately observe under the
microscope, that the cholera bacteria lose their mobiUty, and unite in
clumps. This is spoken of as an "agglutination." Its appearance is indi-
cated by the turbid liquid becoming clear, and a precipitate forming on
the bottom of the vessel. It is interesting to find that a specific reaction
is taking place here, for it is not possible to. detect an influence upon the
cholera bacteria by the serum of an animal which has withstood some other
infection. Thus an animal which has become immune against typhoid
bacteria does not possess a serum which acts upon cholera bacteria, and
conversely the serum of an animal which has been immunized against
cholera does not have the slightest effect upon typhoid bacteria.

The animal organism not .only produces a specific protecting material
against bacteria, but also against their poisons. Thus, the medium on
which diphtheria bacteria have been cultivated shows toxic properties
after the bacteria have been filtered off. Diphtheria toxine acts even in

' R. Pfeiffer: Z. Hygiene, 15, 268 (1894); 20, 217 (1895); Deut. med. Wochsoh. Nos.
7 and 8, pp. 97, 119 (1896). R. Pfeiffer and Kolle: Z. Hygiene, 21, 203 (1896). R.
Pfeiffer and Wassermann: Ibid. 14, 46 (189.3). R. Pfeiffer and Mar.x: Deut. med.
Wochschr. 1898, 47, 489; Z. Hygiene, 27, 272 (1898).

' M. Gruber: Miinchener med. Wochschr. 1896, 206. M. Gruber and H. E. Durham:
nnd. 1896, 285. M. Gruber: Ibid. 1899, 1329. Cf. R. Kraus: Wiener klin. Wochsch.
1897, 32.

OUTLOOK. 681

small doses. By gradually increasing the dose of this poison to an animal
under experiment, we can immunize it, so that it is able to stand relatively
large amounts of it.' We cannot here go into all the developments
arising from these observations, which are so interesting from a biological
standpoint. We can state merely that bacteria yield to the body sub-
stances which we call toxines. They have a harmful effect upon the normal
cell-metabolism. They are, in part, constantly given up by the bacteria,
while to some extent the toxines are retained by the bacteria in their cell
structure. In the latter case these poisons only become active on the
death of the micro-organism. It is questionable whether we are justified
in looking upon these two groups of poisons as being unlike. It is possible
that the eliminated toxines are the end-products of metabolism. It is just
as plausible to believe that we have here a phenomenon which more closely
resembles a secretion process. From this point of view it is easier to under-
stand why the micro-organisms eliminate such highly complicated sub-
stances; while, on the other hand, the conception that the toxines are
end-products of metabolism seems doubtful, because we usually find that
such substances require but little expenditure of energy for their formation;
i.e., they are lower decomposition products. The strictly specific nature of
the toxines also harmonizes more readily with the idea of a typical secretion
process. In such a case we have to deal with products which are analogous
to the ferments. The toxines given off would then correspond to the
"unorganized" ferments; those remaining in the cells, to the "organized"
ferments.

Just as this classification of the ferments is a purely superficial one, and
has nothing to do with their nature and their manner of action, so it is
perfectly possible that the toxines which are given up freely by the cells
and those which are firmly attached to the cells are essentially identical.
Again, the comparison of the toxines and ferments is also superficial, and
should not prejudice us with regard to the toxines. We do not know
anj'thing definite about their nature. They are classed with the proteins,
and justly so, for only to this class of chemical compounds can we
imagine that such complicated bodies belong. For the same reason we
have concluded that the ferments belong to the protein group, being
probably transformation products of the cell-proteins. Just as the fer-
ments exert a specific action, so the bacterial poisons have well-defined
characteristics. We know of poisonous substances which are produced
by highly organized plants and by animals the action of which is quite
similar to that of the toxines. Thus we have ricin from the castor-oil
plant {Ricinus communis), and abrin from the seeds of Jequirity {Ahrus
prccatorius). Both are extremely poisonous, and an immunity may be

' E. Behring: Deut. med. Wochsch. 1890. E. Behring and Kitasato: Ibid. 1890.

682 LECTURE XXX.

established to offset their action.' Snake venom also belong to this class.
The skin and blood of toads likewise contain such poisons, and they are
also found in the garden-spider. The blood of the eel contains a toxine
belonging to this group. Indeed the number of plant and animal poisons
which have been studied is far greater and the poisons are of a more varied
nature than we can attempt to describe. We are not yet ready to discuss
their constitution, and in fact none of these poisons has been obtained in
a perfectly pure state. It is perfectly clear that this fact affects investi-
gation in the whole field of toxines and antitoxines. To-day we cannot
depict, as sharply as we should like, the effect of the toxines and the cause
of the formation of antitoxines. At present we must resort to hypotheses,
and a state of certainty will prevail only when it is found possible to
establish the constitution of at least one of the toxines. From our study
of the ferments, we can readily believe that the antitoxines are similarly
constituted to the toxines. This seems probable from the specific relations
between these two products. It is important as regards the development
of the modern conception of toxines and antitoxines that we should
recognize clearly where the facts end and speculation begins, for nowhere
has this been forgotten more than in this particular field. Perfect clear-
ness in this respect is essential for the sound development of this branch of
knowledge, because one of the most fruitful and most beneficial hypotheses
of the age governs our conception of the nature of toxine action and the
formation of antitoxines. We have in mind the theory to which Paul
Ehrlich refers all the investigations in this field, and which with unexpected
rapidity has been confirmed by observation after observation, and discovery
after discovery.

This hypothesis is quite generally known under the name of Ehrlich's
side-chain theory.- Ehrlich attempted with his theory, which is closely
related to chemical representations, to bridge over the gaps in our inadequate
knowledge concerning the chemical structure of the toxines. He makes
certain assumptions concerning the nature of the active groups. In place
of the chemical composition, he uses certain names which may be replaced
by definite chemical radicals with the advance of our knowledge. Paul
Ehrlich has not only succeeded in correlating by means of his ingenious
theory many processes in this large field which apparently took place side
by side quite independently of one another, but his theory has, moreover.

' Paul Ehrlich: Deut. med. Wochschr. 1891, 976, 1218. Fortschritte d. Medizin,
1897, 41.

' Cf. Rostoski: Zur Kenntnis der Prazipitine. A. Stuber, Wurzburg, 1902. Carl
Oppenheimer: Toxine und Antitoxine. G. Fischer, Jena, 1904. P. T. MiJller: Vor-
lesungen (iber Infektion und Immunitat. G. Fischer, Jena, 1897. Ludwig Aschoff:
Ehrlich's Seitenkettentheorie und ihre Anwendung auf die kiinstlichen Immunisierungs-
prozesse. G. Fischer, Jena, 1902. Paul Romer: Die Ehrlichsche Seitenkettentheorie
und ihre Bedeutung fiir die medizinischen Wissenschaften. A. Holder, Wien, 1904.

OUTLOOK. 683

the great advantage that upon it as a foundation link after link of a tangled
chain of processes has been disentangled, so that as a result we have before
our eyes a continuous picture of separate processes. Problem after problem
has accumulated, and gradually a new structure has been built which
serves to bring under one roof all the various processes which stand in any
relation to the formation of the anti-bodies. The investigations of Ehrlich
appear especially important to us, because they are the first to bridge over
the chasm which has previously been assumed to exist between physio-
logical and pathological processes in the animal kingdom, so that to-day a
sharp line can no longer be drawn between these two fields. Ehrlich has
pointed out that the formation of the anti-bodies stands in direct relation
to the cell-metabolism. In order to make this relation clear, we shall explain
briefly how Ehrlich represents the assimilation of the nutriment by the cell
as taking place. The individual cells are only capable of taking up and
uniting with their structure those substances which correspond to their
entire composition. The substances taken up must fit into the cells.
The protoplasm possesses groups which are chemically active, and these
have a maximum affinity to a certain arrangement of the atoms in the
nutriment, which it unites to the cell-body. Paul Ehrlich calls these groups
side-chains, or receptors. On the basis of this theory we can easily picture
to ourselves why certain cells reject this and that substance, and on
the other hand assimilate other products. One is tempted to deduce a
purely chemical theory for the process of assimilation, though by doing so
we may be making a grave error. We can easily imagine that a chemical
compound, for instance the benzene ring, may carry side-chains, and that
these may enter into reaction with other complexes. A new compound
would result, but such a reaction is, as a rule, complete when this has been
accomplished. The cell, however, behaves in an entirely different manner.
It constantly utilizes material, and must be continually forming new side-
chains, for it is always confronted with the necessity of taking up nutrient
substances ; that is, the new groups must always be present to combine with
the nutriment. It follows from this assumption, that the "side-chains,"
as conceived by Ehrhch, do not correspond to our present idea of a purely
chemical phenomenon. These side-chains are only hypothetical as yet,
and have nothing definite to substantiate their existence. If we assume
that the various cells have differently constituted side-chains, we will then
have reached the idea of the specific nature of the cells. This also permits
us to venture the assumption that the different nutrients are completely
disintegrated during digestion, and are transformed into homogeneous
products in the intestines. Ehrlich's theory is only completely compre-
hensible from this point of view. The specific groups of the cells must
exactly correspond to those of the nutrient materials, the latter being
established only at the time of assimilation.

684 LECTURE XXX.

With the above in mind, let us apply the side-chain theory to the forma-
tion of antitoxines. We have already stated that the bacterial poisons
are probably very closely related to the proteins. We can easily imagine
that they may have atomic groupings very analogous to those of the nutri-
ents, and that, for this reason, they are attached to distinct cells. The
cell immediately loses its ability to assimilate any nutrient material at
those points where any toxines have attached themselves. If the cell has
not been permanently injured by the poison, it will try to repair the damage
by a fresh supply of side-chains. Under these conditions there may be an
over-production of side-chains, to such an extent that they will not all have
room to attach themselves to the protoplasm; they will consequently be
pushed off, and circulate in the blood. We must not forget that these new
side-chains must correspond exactly in their composition to those to which
the toxines have attached themselves. This "first" side-chain must cer-
tainly have had a definite affinity for the toxine before it combined with it,
in its transport in the organism.

Those analogously constituted side-chains which circulate in the blood
must also have the ability of uniting with toxines, thus making them
harmless, before they reach the cells. According to this view, the forma-
tion of antitoxines is not a new process — the free side-chains are nothing
more than antitoxines — but merely a repetition of a normal function of
the cell. It corresponds to the secretion of the individual cells to which
we have repeatedly called attention. It is impossible for us to go into the
details of the facts which go to substantiate EhrUch's assumption. We
only wish to add that it has been shown that definite bacterial poisons, for
instance tetanus poison, enters into combination with tissue-cells, and
that, on the other hand, we are acquainted with poisons which can be recog-
nized as having very distinct aflSnity for specific tissues. Thus, it is
known that abrin possesses very close relationship to the components of
the tissues of the conjunctiva. Ehrlich designates the group of the toxine
molecule, which unites with the side-chains of the cells, or the free side-
chains, as the haptophor group. It is clear, if this conception of the com-
bination of toxines and antitoxines is correct, that only a definite amount
of the latter can combine with a given quantity of the former. The whole
process must evidently correspond to a neutralization.

The toxine also contains a toxophor as well as a haptophor group. This
is the carrier of the specific poisonous effect of the toxine. That this
assumption of different groups in the toxine molecule is well founded fol-
lows from the fact that antitoxines may be produced even after the toxophor
group itself has been destroyed. It has, itself, nothing to do with the im-
munity reaction of the organism. In the latter case the haptophor group
only must be taken into consideration. If this be removed, for instance
by antitoxine, then the toxine will be rendered valueless for immunization.

OUTLOOK. 685

According to this conception, every body-cell must be able to form anti-
toxines, although such an assumption is not absolutely necessary. We
can also imagine that individual cell-complexes possess the ability" of pro-
. ducing toxines; in fact, there seems to be evidence of a selective action
within certain limits. The whole subject of the formation of antitoxines
is analogous to that of the general process of metabolism. We can
easily imagine that the cell-metaboUsm is so altered by the introduction
of toxines that an over-production of side-chains results. This assumption
only serves as an assistant hypothesis, which is to act as a prop to the main
idea which is likewise hypothetical in its nature. It is entirely possible
that the idea of such an enlarged production of atomic groups, and their
expulsion beyond the influence of the cell, is not at all necessary. We
must call attention to a process which we have already discussed in
detail. We have repeatedly remarked how from the carbon dioxide of
the air products of entirely different properties are formed as soon as it
comes in contact with the plant cells containing chlorophyll. They are in
the first place optically active, and contain in their composition hydrogen
as well as carbon and oxygen. We are accustomed to assume that the
first product formed is a carbohydrate, although this beUef has no sub-
stantial foundation. Other compounds, as well as carbohydrates, might
be formed just as easily. Why is an optically active, very specifically
constituted substance, formed from carbon dioxide and water? We are
unable to answer this question. We must assume that this phenomenon
is mainly dependent on the composition of the protoplasm of the chromo-
phyll-containing cells. This is, itself, asymmetrically constituted, and can
consequently only produce asymmetric compounds. When we consider
the question why the cells of the stomach only dehver pepsin or hydro-
chloric acid, and those of the pancreas likewise give a very specific
secretion, we must answer that in this case also the constitution of the
cells is the fundamental cause of the individual functions. Every body-
cell evidently endeavors to maintain its composition, for its permanency
guarantees that its function remains the same and that there is a normal
progress of its metaboHsm. The whole organization of the animal body
is so adjusted that the cells shall maintain their specific composition. This
is already evident from our consideration of the subject of digestion. We
do not in the least doubt that every individual body-cell continually forms
a definite secretion, thus participating in the general metabolism. But
the same food is being normally presented to the cells by the blood.
If a foreign substance passes beyond the intestine, manifold assistance is
offered as quickly as possible all over the organism to forestall any damage.
The liver, especially, guarantees the constant composition of the blood.
It captures material, unites it with other products, etc. If its functions
are not sufficient, other organs come to assist, while the different glands

686 LECTURE XXX.

finally begin to participate in removing the foreign substance by an in-
creased elimination of secretions. The animal organism under normal
circumstances constitutes a well-protected entity. Nothing foreign can
penetrate into the cell-metabolism, consequently the general metabolism
proceeds along its usual course. It becomes an entirely different matter
when material is presented to the cells which can turn the whole organiza-
tion toward an entirely different direction. There are constantly cells in
our body which are engaged in process of destruction, and others, which
here and there renew an important foundation stone, or even entirely
reconstruct it. The body-cells have become adapted to a definite nutri-
tion through many generations, and confine themselves to material which
is useful to the whole organism. During infection, the blood will trans-
port substances which are evidently closely related to normal nutrient
materials.

This assumption seems all the more probable when we suggest that we
can easily imagine how in the preparation of the building-stones of a cell,
not only the cell in question is active, together with its neighboring cells,
but there must be an intimate exchange of the products of metabolism on
the part of the separate cells. Now the toxines are merely products result-
ing from the metabolism of cells. If these products become a part of a
body-cell, then immediately the entire function of such a cell is changed.
It will, as before, receive and give up substances to the fluids of the body,
but the substances now given up will be of an entirely different character,
for the function of a cell is a result of its own composition. We can easily
understand how, if a single constituent of a cell is altered, all the chemical
processes may take place in a different direction. If the cell is not badly
injured it will continue to function. Naturally the subsequent absorp-
tion of material will take place in accordance with the altered conditions,
and thus the peculiar nature of such cells will gradually make itself felt.
Among the thousands of body-cells, it is not necessary that many of them
should be attacked, perhaps only those which were in the process of under-
going a transformation. We know that in chemical processes the slightest
deviation in the conditions may cause the reaction to take place differently.
How much more must a continuous change in the nature of the metaboUc
products affect the normal course of processes which take part in the cell
construction! We wish to affihate this conception of Ehrlich concerning
the formation of antitoxines more closely with processes of metabolism,
and especially in order to avoid leaving the impression that the formation
of these side-chains is an abnormal function of the cells. The different
toxines are naturally differently constituted, and it does not seem at all
strange that the cells of the various tissues should take up these to.xines
in different degree, and that, for instance, the cells of the nerve tissues
should be peculiarly adapted to unite with tetanus poison. This toxine

OUTLOOK. 687

shows very clearly that Ehrlich is right in his conception of the production
of toxines by the cells. Wassermann and Takaki ' carried out the fol-
lowing experiment. They triturated the spinal cords and brains of
normal guinea pigs with a physiological salt solution. Into this emulsion
they introduced a single, double, treble, and ten times deadly dose of
tetanus poison and injected these mixtures subcutaneously into mice.
The animals did not die. It is noteworthy that this antitoxic action of
tetanus poison is confined solely to the nerve tissues. It was also shown
that the brain and spinal cord emulsions likewise act antitoxically if they
are injected subcutaneously into mice and then the various lethal doses
afterward administered. Similarly the poisonous effect is much lessened if
the emulsions in question are introduced after the toxine into the body.
It is possible that we shall be able to isolate the poisonous group in tetanus
poison and find out its composition. Centrifugalized emulsions of nerve
tissue are perfectly inactive, which proves that we are not concerned with
the substances in the surrounding fluids but with the cells themselves.
The discovery that the antitoxine from the brain is destroyed by boiling,
and that the protective effect of the emulsion obtained from the spinal
medulla or brain is lost in the same way, is of great significance. Blumen-
thal" has at last succeeded in proving that the tetanus poison combines
with the brain substance, by adding the tetanus poison, which itself can
readily pass through a filter, to brain substance and then filtering. The
filtrate contains no toxine. It might be thought that perhaps the solids
of the nervoas tissue had held it back merely mechanically. That this is
not the case is shown by the fact that the mixture of tetanotoxine and
nerve substance no longer has a toxic effect. Finally Blumenthal suc-
ceeded in proving that the protective action of the brain and cord grew
less in proportion to the amount of toxine which had been administered
to the animal in Hfe. It is easy to explain why this is true. The brain
substance cannot, of course, combine with an infinite amount of toxine.
The amount taken up naturally depends upon the number of the reacting
groups that are present which have an affinity to tetanotoxine. If some
of these groups have already been satisfied, then naturally this tissue
will be capable of removing only a fraction of the amount which it is
otherwise capable of uniting with. In fact, we can determine quantita-
tively how much antitoxine is present in a certain amount of nerve sub-
stance by estimating the point at which it ceases to combine with more
toxine.

We cannot here go into further details concerning the toxines and anti-
toxines, nor discuss the development of Ehrlich 's side-chain theory in
this direction anv further. It is sufficient for us to have sketched the

' Wassermann and T. Takaki: Berliner klin. Wochenschrift, 1, 5, 1898.
> F. Blumenthal: Deut. med. Wochschr. No. 12, p. 185 (1898).

688 LECTURE XXX.

extent of the investigation in this field and to have shown how closely
related it is. to physiological conceptions. We shall now turn our attention
to the results of Ehrlich's hypothesis in the study of hemolysis, which is
more closely related to our subject.

It is a well-known fact that the sera of many varieties of blood are able
to dissolve the red corpuscles of other species of animals. This fact is the
cause of the bad results which have resulted from the attempts at the trans-
fusion of blood into human beings. For a long time little was known
concerning the nature of hemolysis. Recently Belfanti and Carbone '
have estaljlished the fact that the serum of a horse into which the red cor-
puscles from rabbits have been injected, has a much more poisonous effect
upon rabbits than does normal horse serum. Bordet,- whom we have to
thank for developing our conception of hemolysis, showed that the serum
of guinea pigs into which there had been repeated intraperitoneal injections
of from three to five cubic centimeters of defibrinated rabbit's blood, would,
when placed in a test-tube, rapidly dissolve the red corpuscles of the rabbit,
whereas the normal serunxof the guinea pig either did not have this property
at all, or showed but slight evidence of it. Here again we are dealing with
quite specific effects, and there is really a formation of anti-bodies here.
This discovery is of especial interest because Bordet has shown that even
cells to which we are not accustomed to ascribe toxic effects have a quite
similar effect to that of the bacteria. The phenomenon is not remarkable.
It merely shows us that every species of animal has its own peculiarly
constituted cells and thereby its own specific metabolism.

We must, first of all, consider the explanation of how the hemoglobin
is removed from the red corpuscles under the action of the serum which
is employed. It cannot be a question of variations in the osmotic pres-
sure, for even a fraction of a milligram of the serum exerts this effect, and
again the specific action also contradicts any such assumption. All of our
knowledge points to a poisonous effect upon the red corpuscles them-
selves. We shall now try to apply Ehrlich's side-chain theory, as far as
possible, to the phenomenon of hemolysis. Bordet succeeded in proving
that a hemolytic serum loses its effect if it is heated to 55° C. The
serum is then designated as inactive serum. In order to avoid any mis-
conceptions, we had best take up a specific example. Let us assume that
we have a guinea pig which has previously been treated with rabbit's
blood. If the serum of this animal is allowed to fall drop by drop upon
an opaque solution of red corpuscles from a rabbit, contained in isotonic

• S. Belfanti and P. Carbone: Giom. della R. Accad. di med. di Torino, 1898.

' J. Bordet: Ann. inst. Pasteur, 12, 688 (1898) ; 13, 273 (1899) ; 14, 257 (1900) ; 15, 303
(1901). Cf. Von Dungern: Miinchener med. Wochschr. Nos. 13 and 14, pp. 405, 449
(1899). K. Landsteiner: Zentr. Bacteriol. 25 (1899). P. Ehrlich and J. Morgenroth:
Berliner klin. Wochschr. 1899, 1900. 1901. P. Ehrlich and H. Sachs: 1902.

OUTLOOK. 689

salt solution, we shall find that within a short time a solution of the red
blood-corpuscles takes place. The solution becomes transparent and a
clear red. If exactly the same experiment is carried out with inactive
serum, the red corpuscles will remain unaffected. Now if in a third test-
tube the serum of a normal guinea pig is added to the suspension of
blood-corpuscles, there is again no hemolysis. On the other hand, the
solvent effect is obtained if the normal serum from normal guinea pigs is
added to the inactivated serum. This proves that at least two substances
are necessary for bringing about hemolysis. One of these is found
already formed in the immune serum and also in normal serum, and the
other is only yielded by immunized serum, i.e., in this case in the blood
of guinea pigs which have been previously treated with the blood
of rabbits. The two substances are different as regards their behavior
toward heat. That which is present in normal serum is stable towards
heat, while that present in the other is unstable. Quite a number of different
names have been given to these substances. We shall choose for the
thermo-stable substance the designation amboceptor, and for the thermo-
unstable one the name complement.

Ehrlich's theory fits in at this point. He was especially interested in
explaining why the different sera should act so specifically, and what the
relations of amboceptor and complement are to the red blood-corpuscles
and to themselves. For simplicity's sake we will designate the two com-
ponents, amboceptor and complement, which produce hemoly-^is, by the
name hemolysin. This must have, judging from its analogy to the to.xines,
a very distinct affinity to some constituent of the red corpuscles. Ehrlich
and Morgeriroth's experiments have proved this. They injected sheep's
blood into a goat. The serum from this animal eompletely dis.solved the
sheep-blood corpuscles, although it lost this property on heating to 55
degrees. The complements were destroj'ed. The amboceptors must
■have remained unchanged, as they are thermo-stable at this temperature.
The above investigators then added sheep-blood corpuscles, and centri-
fugalized the mixture after standing half an hour. To the resulting serum
they added more sheep-blood corpuscles, and also some fresh normal
serum. No hemolysis resulted. When the previous addition of sheep-
blood corpuscles was omitted, and normal serum added to the inactivated
serum, the solution of the red corpuscles immediately set in. It follows
from these experiments that the sheep-blood corpuscles had removed one
of the components of the hemolysin which was, in fact, the amboceptor.
That this assumption is correct, is evident from the fact that the blood-
corpuscles, centrifugalized as above, immediately went into solution on
the addition of normal serum in an 0.85 per cent salt solution. We must
also mention that normal goat's-blood serum does not attack the normal
sheep-blood corpuscles.

690 LECTURE XXX.

One phase of hemolysis was, therefore, explained. The amboceptors
present in immune serum unite with the red blood-corpuscles of that species
of animals to which they are suited. No other kind of blood is able
to combine with the amboceptors which react so readily with sheep's
blood. For this reason alone we cannot assume that it is a case of mere
absorption of the blood amboceptors by the red blood-corpuscles. The
combined amboceptors cannot be removed by washing, and in fact the
affinity of these for the red corpuscles may be determined. It has been
found that different red corpuscles are capable of combining with very
different amounts of amboceptors.

We must now attempt to explain how the complement stands in relation
to the process of hemolysis. In the first place, Ehrlich and Morgenroth
have proved that normal red corpuscles do not unite with the complements.
The simplest explanation is that the amboceptors possess at least two
differently constituted groups. One unites with the blood-cell, and the
other with the complement. This effects the solution of the blood-cor-
puscles. The complement alone cannot act upon them. The groups
which are adapted to act upon the red corpuscles are wanting. Only by
the aid of the amboceptor is the complement able to react with the erythro-
cytes. Just what this influence is we cannot tell, but it is possible that a
fermentation takes place. We may state that hemolysin has been con-
sidered to be analogous to the toxines. It is in a sense a compound
toxine. The haptophor group of the toxine corresponds to the ambo-
ceptor, and the toxophor group to the complement. The comparison
seems even more justifiable when we add that it has been found possible
to form anti-bodies to the hemolysins. Certain poisons of the animal
and vegetable kingdom are analogous to the hemolysins.' We may
mention snake venom, garden-spider poison (Arachnolysin), and toad
poison iPhrxjnolysin) . It is also possible to obtain poisons with a hemo-
lytic action from bacterial cultures. We may refer to stapholysin which
is obtained from staphylococcus cultures, and tetanolysin from tetanus
bacteria.

Here at this point we may also refer to an observation which we have
already discussed in detail.^ One of the many poisonous effects of snake
venom is its hemolytic action. If, for example, we add the poison of the
cobra to blood, hemolysis soon sets in. If the blood-corpuscles, how-
ever, are well washed, i.e. freed as completely as possible from every trace
of serum, and then placed in 0.85 per cent sodium chloride solution, no

' Cf. Flexner and Noguchi: J. exper. Med. 6, No. 3 (1902). H. Sachs: Hofmeister's
Beitr. 2, 125 (1902). F. Proscher: Ibid. 1, 575 (1901). R. Kraus and P. Clairmont:
Wiener klin. Wochschr. 1900, No. 3, and 1901, 1016. M. Neisser and F. Wechsburg:
Munchener med. Wochschr. 48, No. 18, p. 097 (1901) and Z. Hygiene, 36, 299 (1901).

' Cf. Lecture VI, p. 115, el seq.

OUTLOOK. 691

hemolysis takes place on the addition of cobra poison. The addition of
a little serum, or of lecithin, now suffices to cause hemolysis. It is natural
to designate lecithin as the amboceptor which enables the poison of the
cobra to attack the erythrocytes. It is, furthermore, interesting to find
that cholesterol can prevent this action of the lecithin. We mention
these interesting discoveries here because they perhaps explain why the
two compounds, cholesterol and lecithin, are found in every cell.

There is no doubt that red blood-corpuscles are constantly being de-
stroyed in our organisms. Hemolysis undoubtedly takes place. It may
be brought about by changes in the osmotic pressure, but it is also possible
that the normal destruction of the red corpuscles results from a process
similar to that just described.

We may add that the formation of precipitins may also be explained on
the basis of Ehrlich's side-chain theory, and many other discoveries find
their proper place in complicated processes by means of the same theory.

We have now reached the end. We are aware that we have just placed
considerable stress upon a mere hypothesis, and realize the danger that may
result from such a generalization. On the other hand, we are able, in each
particular case, to decide whether we are justified in comparing a given
process with those which have been just described. It is clear that the
final goal of our investigation in the field of immunization is never to be
attained by the mere advancement of any special theory. We find our-
selves temporarily in a region which is not accessible to chemical or
physical investigation, for both require as a starting-point the employment
of chemically pure substances. As long as we are unable to prepare any
one of these complicated products in a pure state, and establish its
chemical constitution, we must not expect to obtain an exact insight into
all the complicated processes upon which the actions of the toxines and
related substances are based.

AUTHOR INDEX.

Abderhalden, Emil, protein hydrolysis,
126, 134, 172-176; partial hydrolysis of
silk, 186; fermentation hydrolysis, 152,
166, 204; pepsin digestion, 165, 192;
trypsin digestion, 165-167; purpose of
digestion, 61, 110, 579, 638; formation
and decomposition of protein, 100, 164,
204, 209, 212, 245, 304; di-amino-tri-
hydroxydodecoic acid, 154, 318; cystine,
159; carbohydrate group, 126, 161; pro-
tein derivative in urine, 49, 269, 270;
Bence-Jones albumin, 269; albumin
synthesis in the animal body, 213, 224,
355; protein requirement, 640; Asper-
gillus niger, 216; cystinuria, 267, 268;
alcaptonuria, 273; phosphorus poison-
ing, 264; decomposition of polypeptides,
184, 185, 192, 228. 249, 474; proteolytic
ferments, 192, 506; decomposition of
racemic amino acids, 452; amino acids
in urine, 264, 266 ; peptones in blood,
211; cholesterol, 117; hemolysis, 115;
hemoglobin, 125, 126, 382, 558 ; analysis
of blood, 114, 116, 365, 368, 553, 556,
666; effect of high altitudes on blood,
435; sodium chloride (table salt), 364;
the iron question, 382, 390, 392, 394;
milk, 366, 654, 665; casein, 171, 656;
rate of development, 371, 404; ash of
sucklings, 368; oxidation by microbes,
448; parthogenesis, 360, 672; adrenalin,
601 ; nucleic acids, 288; hemophilia, 543;
conception of species, 664, 666.

Abel, J. John, carbamic acid, 232, 602.

Abeles, M., 92.

Abelmann, 106.

Abelous, J. E., hippuric acid, 247;
oxydases, 447.

Addison, T., 603.

Aders-Pli.mmer, R. H., gelatin, 176;
lactase, 323.

Adler, O., 29.

Abler, R., 29.

Aeby, 428.

AroNASsiEW, 409.

Albert, R., 464.

Albertoni, p., acetone, 99; coagulation
of blood, 546.

Aldehoff, 83.

Alfthan, K. v., 48.

Alsberg, C, 266.

Andre, 333.

Andre, 586.

Anten, Henri, 586.

Araki, T., metabolism with insufficient

oxygen, 75, 237; phosphorus, arsenic,

etc., poisoning, 81 ;hydroxy-butyric acid,

97; blood pigment, 561.
Armstrong, F. E., sugar, 38; syntheses by

ferments, 4S0.
Arn.schink, 105.
Aron, H., 361.
Arthus, diasta.se, 71 ; glucolysis, 87; blood

coagulation, 536, 538.
AscHOFF, L., 682.
A.SCOLI, A., 281.
AsHER, L., blood-sugar, 30, 587; lymph,

576.

ASTASCHEWSKY, 75.

Athansiu, J., 328.

Atterberg, a., 306.

Atwater, W. O., metabolism, 335, 338,

339, 340, 344, 345, 346, 622, 623, 626,

646, 651.
Aubert, H., 437.
Autenrieth, W., 444, 457.
AuvERS, K., 15.

B.

B.A.AS, K. H., 615.

Bab.^k, E., 438.

Babkin, B., legumins, 173; breaking-down
of polypeptides, 228; pancreatic juice,
522, 526, 530.

Bach, A., oxidation ferments, 54, 449;
carbonic acid assimilation, 57, 201.

Baeyer, Adolf v., carbonic acid assimila-
tion, 15, 56; uric acid, 278.

Baglioni, S., 225.

Baisch, K., 48.

Baldi, 49.

Balke, 298.

Bamberger, M., 200.

Bang, Iv.\r, guanylic acid, 22, 284;
albumin, 135, 139; parachymosin, 207.

Bar, p., 642.

Barbera, a. G., salivary glands, 486;
bile, 513; lymph, 576.

Barbieri, J., amides, 151; phenyl-
alanine, 151; allantoine, 296. \

Barcroft, J. L., 410, 584.

Barfurth, 351.

Barfurth, Dietrich, glycogen, 46.

Barker, L. F., 264.

Barral, 73, 87.

Barreswil, 44.

Barth, H., 444, 457.

694

AUTHOR INDEX.

Bartolleti, F., 13, 39.

Bahy, de, 53.

Bashford, E., 247.

Batelli, F., 449.

Baumann, E., mercapturic acid, 158;
putrefaction of protein, 169; ethereal
sulphuric acids, 243, 250, 251, 252, 253,
254, 457, 458; crystine, 266; cystinuria,
266 ; alcaptonuria, 271 ; iodothyrin, 607.

Baumert, 438.

Badmgarten, 0., 94.

Baumstakk, F., 613.

Bayer, H., plaste'm, 208; o.xygen require-
ment, 615.

BAYLIS.S, W. M., secretin, 168, 527; lymph,
574.

BeaUiMont, W., 203.

Bechold, H., 677.

Beddard, a. p., 582.

Behring, E., 681.

Beijerinck, M., 53, 197, 217.

Belfanti, S., 688.

Bendix, E., 22, 294.

Benech, B., 156.

Berdez, J., 145.

Bergell, p., choline, 113; carbohydrate
group of proteins, 126, 161; breaking-
down of polypeptides, 184, 185, 474;
phosphorus poisoning, 264; adrenalin,
601.

Bergmann, Tobern, 277.

Bergm.inn, v., 248.

Berminzone, M. R., 247.

Bernard, Claude, glycogen, 44, 45, 67;
diastatic ferments, 478; glucosuria, 38,
73, 81 ; diabetic puncture, 76; formation
of glycogen, 319; color of blood in
glands, 486.

Bernhardt, M., 77.

Bert, Paul, 417, 435.

Bertagnini, 243.

Bertel, R., 202, 273.

Berthelot, lutrogen assimilation, 195;
calorimetry, 333; invertin, 461.

Bertrand, G., lactase, 447, 450; sorbose,
448 ; arsenic, 407.

Bial, M., pentosuria, 23; diastase, 73.

Biarnes, 447.

Biberfeld, J., 584.

Bickel, a., 499.

Bidder, 203.

Biedermann, W., 123, 447.

Bienstock, 218.

Bigelow, S. L., 468.

Bing, B., 49.

Bing, Robert, 617.

Biot, polarization, 15.

Biot, 463.

Blackman, F. F., 51.

Blendermann, H., 255.

Blum, L., 249.

Blumenreic^h, L., 611.

Blumenthal, F., pentoses, 21, 23; tetanus
poison, 685.

BocKEL, K., 177.

Bodong, a., 547.

Bodeker, 270.

BoHM, 30, 72.

Bottcher, 25.

Bogdanow, E., 325.

Bohr, Christian, respiratory exchange,
415, 417, 419, 425, 426, 427, 430, 431,
432, 4.33, 435, 441; hemoglobin, 560.

BOKORNY, T,, 357.

Boldiereff, 512.

BoNDZYNSKi, St., 117, 269.

Bontroux, 448.

BoRDET, Jules, 545, 668, 688.

Bornstein, Karl, 642.

Boruttau, 602.

bouchardat, 39, 91.

BouiLLAC, R., 57.

BouLUD, 32.

Bourquelot, E., 447, 449.

BoUSSINGAULT, 56.

Br.^connot, H., 148.

Brahm, C, 252.

Brandenburg, K., 422.

Brandt, K., 198.

Bredig, G., 467, 468.

Brieger, 169, 251, 458.

Brion, a., 452.

Broder.sen, 398.

Brodie, T. G., 584, 617.

Brown, 477.

Brown, H. P., 42, 59.

Brown-Sequard, E., 600.

Brucke, 545.

Brunner, Arnold, 173.

Bruyn, C. a. Lobry de, 67.

Buchanan, 5.36.

Buchner, E., 448, 463, 465, 482.

Buchner, H., 463.

Bugarsky, S., 120.

Bulnheim, 515.

Bulnheim, G. v., hippuric acid, 9, 246;
oxygen requirement, 74, 443; table salt,
354, 362, 363, 366; ash of suckUng, 368;
iron, 368, 380, 381, .384, 387, 399, 401;
lime, 370, 372, 377; blood, 553; lymph
fonnation, 575; composition of foods,
648; vegetarianism, 649; biogenetic law,
673.

Bunte, Karl, 277.

burckhardt, a., 556.

Burian, Richard, uric acid, 10, 241 ;
nucleic acid, 282, 284, 285; purine
metabolism, 287, 291; purine bases, 290,
291 292.

Bu.sch,"'f. W., 576.

Bussy, 461.

Byk, A., 55.

Camerer, W., 290, 368, 593, 655.

Cantani, a., 94.

Carbone, p., 688. -

Carvallo, G., 509.

Cash, 104.

Caspari, W., 436, 643, 649, 651.

AUTHOR INDEX.

695

Cavazzani, E., 72.
Centnerswer, M., 468.
Chandelon, T., 68.
Chaniewski, St., 309.
Chauveau, a., 338.
Chevalier, J., 614.
Chevreul, 90, 101.
Chigix, p., 502.
Chod.\t, R., 54, 449.
Chossat, T., 375.

ClENKOWSKI, 533.

Cl.virmont, 690.

Claus, R., 89.

Clautri.an, 47.

Clemens, P., 34.

Clerget, 15.

Cleve, p. T., 515.

Closson, O. E., 587.

Clowes, G. H. A., 374.

CoHN, Rudolph, leucinimide, 169; conju-
gated compounds, 234, 243, 244, 245,
246, 457; sugar formation, 318.

CoHN, Theodor, 296.

Cohnhei.m, OiTO, carbohydrate combus-
tion, 89; albumin, 131, 134;erepsin, 167,
209; work of glands, 341; mountain
sickness, 436 ; absorption in small inte?-
tine, 532.

CoLB, S. W., 152.

CoLUCCi, V. L., 674.

CoNN'.STEiN, W., lipase, 103; fat absorption,
105; lipolytic action of blood, 109;
lymph, 574.

CORANDA, 2.30.
CORVISART, 164.
CoURMONT, 586.

Cr.amer, a., 47. '

CR.4.MER, E., 149.

Cra-mer, W., 247.

Cr.\wford, a., 601.

Cremer, Max, 66, 81, 83, 314.

Cz.^PEK, F., 51, 103, 118, 216, 273.

Czerny, 509.

CZERNY, F., 73.

D.
D.4KIN, H. D., 1.35, 137, 175, 226, 475.
D.\M.\SKiN, N., 526.
Danilewsky, a., 208.
Dapper, Carl, 290.
Dastre, a., 107, 547.
Dauxay, R., 642.
Davy, H., 193.

Delezen.ve, C, 539, 541, 547.
Denis, 537.
Despretz, 335.
Dhere, C, 402.
Diakonow, 112.
Di.amare, v., 83, 90, 225.
Diels, Otto, 117.
Dietschy, R., 230, 211.
DiTTHORN, Fritz, 20.
Dock, F. W., 77.
DORPINH.ATJS, T., 126, 161, 17&
Do.MBROwsKi, St., 269.

dominicis, de, 87.
Dor.meyer, C, 325.
DoYON, 548.

Drechsel, E., jecorin, 49; albumins, 123;
lysine, 154 ; urea, 226 ; carbamic acid, 232.
Dreser, H., 583.
Dubrunf.\ut, 38, 39.
DuLONG, 335.
Dungern, v., 688.
DUPETIT, 195.
DuRH.\M, H. E., 680.
Durig, a. E., 436.

E.

Eberle, 203.

Ebner, V. v., 123.

Ebstbin, E., 22.

Ebstein, W., cystinuria, 266; elimination
of uric acid, 586.

Eckh-ard, C, 78, 80.

Edinger, L., 616.

Ehenstein, W. Alberda van, 67.

Ehrlich, Felix, isoleucine, 148; cleavage
of racemic bodies, 470.

Ehrlich, Paul, oxidation, 411, 431;
chemical constitution and disinfecting
effect, 677; side-chain theory, 682-691.

Eiselsberg, a. Freih v., 607.

Ellinger, Alexander, tryptophane, 153,
258; cadaverine, 154, 260; putrescine,
155, 260; intestinal putrefaction, 220;
kynurenic acid, 260; Bence-Jones pro-
tein, 269; lymph, 574.

Embden, G., 89, 264, 273, 319, 454.

Emerson, R. C, 259.

E.MMERLiNG, O., isomaltose, 37; cystine,
157; papayotin, 168; amino acids as
nutriment for molds, 216; oxidizing
molds, 448.

Engelmann, T. W., chromophyll, 52, 53;
heredity of acquired properties, 672.

Engler, C, 449.

Erdmann, J., 42.

Erlen.meyer, Jr., 158.

Erman, 326.

Ernst, 468.

Errera, 47.

e.scherich, 218.

Eschle, 215.

EuLER, Astrid, 57.

EuLER, Hans, 57.

Ewald, C. a., 105.

F.tLK, E., 630.
Falta, W., 271, 273, 355.
Fang, 546.
Farkas, G., 549.
Feder, L., 230.
Feer, E., 221.
Fehling, H., 378.
FicK, A., 69, 412.

FlECHTER, 464.

Filehne, W., 584.

FiLippi, Filifpo de, .389, 402.

69G

AUTHOR INDEX.

Fischer, Emil, carbohydrates, 13-17, 19,
20, 26, 27, 31, 32, 35, 37, 38, 55, 58;
protein, amino acids, serine, 149; pro-
line, 151; diamino-trihydroxy-dodecoic
acid, 154, 318; lysine, 155; ornithine,
156; cystine, 159, 266; breaking down
of proteins, 149, 152, 171, 174, 176;
partial hydrolysis of silk, 187; breaking
down of polypeptides, 178-187; fer-
mentation of proteins, 152, 165, 166,
204; of polypeptides, 184, 185, 192, 474;
uric acid and purine bases, 277, 278,
279, 281 ; physiological significance of
stereochemistry, 471 ; fermentation of
sugar, 471 et seq.; sense of smell, 494;
taste of the amino acids, 494.

Fischer, Martin H., 80, 359.

FiscHL, R., 610.

Flammand, 153.

Flechsig, E., 62.

Fleissig, p., 56.

Fleitmann, 157.

Flexner, S., 115, 116, 690.

Floresco, 547.

Foa, p., 540.

Foges, 600.

FoLiN, Otto, 266, 588.

FONTANA, 547.

FOR.STER, 354, 375.

fourcroy, 277.

Fraenkel, a., 435.

Fraenkel, p., 549.

Fraenkel, S., 271, 677.

Framm, F., 73.

Franchimont, 42.

Frank, Otto, 105, 325, 326.

Frankfurt, S., 38.

Franz, 547.

FuEDERicQ, L., 402, 421, 431, 537.

Frentzel, J., 70, 123, 312.

Frerichs, 236, 242.

Frerichs, F. T., 92, 99.

Freund, 545.

Frey, Max v., 70.

Friedel, J., 54.

Friedemann, U., 667.

Friedenthal, H., diastase, 60; fat absorp-
tion, 108; biological reaction, 670.

Friedleben, a., 010.

Friedmann, E., cystine, 157, 158; mer-
eapturic acid, 159; thiolactic acid, 169.

Frisbie, W. S., 374.

Frohlich, 615.

Fromm, Emil, 34, 35, 244, 458, 678.

Fromme, Albert, 104.

FtJRTH, Otto v., proteins, 146; muscle
protein, 133, 617, 618; tyrosinase, 447;
comparative physiology, 402.

FuLD, 547.

G.

Gabriel S., 215, 403.
Gad, J., 103.

Gaidukow, N., chromophyll, 52; heredity,
672.

Gamgee, 203.

Garnier, L., 273.

G.\RROD, A. E., alcaptonuria, 272 ; chemical

anomalies, 675.
Gatin-Gruzewska, Z., 45.
Gaule, J., 390.
Gautier, a., 407.
Gayon, 195.
Geelmuyden, H. C, 97.
Gelpke, L., 377.
Generali, 607.
Gengou, 545.
Gentzen, Max, 258.
Geppert, J.', 413, 435.
Gerber, C, 307.
Gerhardt, C, 97.
Gbrhardt, D., 218.
Gbrngrcss, Otto, 281.
GiACOSA, P., 243, 289.
Gierke, E., 46.
GiES, W. J., 576.
Gilbert, R. D., 653.
GiLsoN, E., 112.
GiRARD, H., 72.
GlUSTINIANI, 57.

GizELT, A., 527.

Glassner, K., 505.

Glauber, 13.

Gley, E., 606.

godlewski, e., 672.

Goldman, E., 249, 266.

goldthwait, 378.

gonnerm.a.nn, m., 273, 447.

Goto, M., 136.

Gottlieb, R., 269, 389.

Graebe, 21.

Grate, V., 42.

Graham, T.. 120.

Green, J. Reynolds, 202.

Griesmayer, v., 131.

Griffiths, 402.

Grube, K., 66.

Grube, Max, 680.

Griitzner, p., gastric digestion, 60, 500;

urea formation, 583.
Grund, G., 22.
gscheidlen, r., 2.50.
GiJRBER, A., albumin, 124; alkali of the

blood, 426.

GtjRTLER, F., 81.

Gulewitsch, W., thymine, 281.
Gull, W., 605.
Gundelach, C, 514.
GuRWiTSCH, A., 582.
Gusserow, a., 295.

H.

Hatjsermann, Emil, 380.
H.\HN, M., 231.
Hahn, Martin, 463.
Halban, J., 599.
Haldane, John, 431.
Hall, Walker, 285.
Hall, W. S., 389.
Hallervorden, E., 230.

AUTHOR INDEX.

697

Halliburton, W. B., 614.
Hamburger, 60.
Hamburger, .389.
Hamburger, Fr.\nz, 664, 668.
Hamburger, H. J., 361, 426; blood, 532,

550; lymph, 574.
Hammar, J. A., 610.
Hammarsten, Olof, nucleoproteids, 21

134; mucins and mucoids, 142, 144

rennin, 205; bile acids, 225, 515, 516

blood coagulation, 537, 538.
Hannon, 401.
Hansen, 355.
Hansen, A., 51.
Harden, \., 47.
Hardy, W. B., .361.
Harless, 402.
Harley, v., 73.
H.\rnack, E., muscarine, 114; albumin,

128, sulphhemoglobin, 561.
Harries, C, 305, 312.
Harris, Isa.\c F., 23, 139, 285.
Harroy, M., 54.
Hart, Edwin, 174, 177.
Hartig, T., 122.
Harkins, H. D., 233.
H.asselb.\ch, K., 427.
Haycr.\ft, John, levulose, 67, 95; hirudin,

547.
Hedon, E., 77, 83.
Heffter, a., 75.
Heidenhain, Martin, 5.32.
Heidenhain, Rudolf, salivary glands, 79,

486, 487; fat absorption, 106; lymph

formation, 547, 574; urine secretion,

582.
Heinemann, H. N., 312.
Heintz, 515.
Hele, T. S., 272.
Hellriegel, H., 196.
Hen,neberg, W., 62.
Henriques, 355.
Henriques, v., 431, 441.
Hefner, E., 116.
Her.\eus, W., 193.
Hermann, 74.
Hermann, A., 352.
Her.mann, L., 561.
Heron, John, 42, 60.
Herrik, J. B., 173.
Herrm.\nn, E., 407.
Herter, C. a., 411, 615.
Herter, E., 243, 252, 457.
Hertwig, Oskar, 53.
Herzog, R. O., 54.
Heubner, Otto, 655.
Hilbert, Paul, 458.

Hildebr.\ndt, Hermann, decomposition
of cyclic compounds, 32, 34; ferments,
476.
Hill, Artur Croft, 37.
HiRSCH, A., 507.
His, 351.

His, W. d. J., 298, 593.
HOCHHAUS, 389.

HoBBER, Rudolf, 5.32, 591 ; effect of ions,
361; blood, 549; kidneys, 587.

Hoessli, \., 543.

Hoff, J. H. van't, 15, 16, 25.

Hoffmann, A., 247.

Hoffmann, F. A., 30, 72.

HoFMANN, A., 389, 395.

HoPMANN, Franz, 109, 326.

Hofmelster, Franz, starvation glucosuria,
30, 91, 95; lactosuria, 39; assimilation
limit for carbohydrates, 76; cell fer-
ments, 88; albumins, 124, 131; albumin
absorption, 208 ; urea, 230.

Hopmeister, Viktor, 62.

HOLLE, 56.

Hopkins, F. G., albumin, 124; trypto-
phane, 152.

Hoppe-Seyler, Felix, cellulose fermen-
tation, 62; metabolism with insufficient
oxygen, 75, 81, 237; respiratory ex-
change, 623; hemoglobin, 125, 424,
559, 561; ozone, 442; blood, 553; urea,
230.

Hoppe-Seyler, G., 398.

Horab.aczewski, J., 10, 278, 289, 290.

Hornborg, 499.

Hoyer, E., 103.

Huber, 71.

HiJFNER, G., hemoglobin, 125, 559, 560,
561, 562; nitric-o.xide hemoglobin, 561;
carbonic-o.xide hemoglobin, 424, 561 ;
oxygen combination, 414, 419.

Hunefeld, F. L., 124.

Hueppe, F., nitrifying bacteria, 193;
vegetarianism, 649.

HiJRTHLE, 608.

HURTHLE, K., 116.

Hugonnenq, L., ash of a suckling, 369;
absorption of salt by the fcetus, 375,
385; hematogen, 387.

HuiSK.\MP, W., histone, 139; fibrin-
globulin, 542.

HuMNicKi, v., 117.

Humphry, L., 609.

Hundeshagen, Franz, 112.

Hunter, Andrew, 671.

HuppERT, 664,

Ikeda, K., 468.
Ingenhousz, 51.
Inoko, Y., 285.
IS.VAC, S., 667.

J.\CKSON, Henry, 202.

J.\cobj, 547.

Jacobj, Carl, 92.

Jacoby, M.\rtin, oxydases, 447; spleen,

611.
Jaderholm, Axel, 125, 562.
J-U^FE, Max, conjugated acids in urine,

34, 457, 458; glycogen, 92; ormithine,

155; indican, 220, 258.

698

AUTHOR INDEX.

Jakohsthal, H., 326.

Jaksch, R. v., 97.

Jaquet, Alfred, carbonic acid combina-
tion, 421; effect of high altitudes, 436,
643; oxydases, 445; hemoglobin, 559.

Ja.STI!OWITZ, 21.

Jawein, Geohg, Gil.
Jeunek, John, 73.
JOHANNSON, J. E., 631, 647.
Johnson, Tre.\t B., 281.
Johnston, H. M., 610.
JoLiN, Severin, 515.
Jones, W., 281.

JOTEVKO, 459.

K.
Kalanthar, a., 473.
Kalberlah, F., 454.
Kaltenbach, p., 39.
Kareff, N., 548.
Kassowitz, M., 309.
Kastle, 446.

K.'iTZENSTEIN, A., 241, 4.52.
Kauffmann, M., 215, 290.
Kauf.mann, M., 634.
Kausch, W., 83.
Kekule, 45.
Keller, W., 242.
Kellnbr, O., 70, 215.
KiESEL, K., 168.
Kiliani, 13, 18.
Kirchhoff, C, 13, 39, 461.
Kirk, 271.

KiSCHENSKY, 106.

KiTAS.ATO, 681.

Kletzinsky, 401.

Knieriem, W. v., celhilose, 62, 63; urea,

228, 230; uric acid, 237.
Knoop, F., 286, 454.
Kobert, R., 388.
KocHER, T., 605.
Kochs, 247.
KoENiG, 376, 645, 646, 648, 651, 654, 655,

659, 661.
KoENiGS, E., 183.

KONIGSBERG, A., 587.

KOEPPE, H., 492, 550.

KOLLE, 680.

KossEL, A., 22; proteins, 134, 135, 137,
173, 175, 176; histidine, 154; arginase,
226; nucleic acids, 22, 277, 258; uracil,
281; thymine, 281; cytosine, 281; for-
mation of purine bases, 287; gas pump,
413.

KowALEWSKi, Katharina, 169, 238.

Kratter, J., 326.

Kraus, 421.

Kraus, F., 326, 328.

Kraus, Rudolf, 680, 690.

Kr.awkow, N. p., 49.

Krieger, H. T., 124.

Krogh, 417, 427, 432, 437.

Kronecker, 459.

KntJGER, 195.

Kkvqer, Albert, 157.

Krugeb, F., 125, 559.

Kruger, M., choline, 113; purine bases of

the fffices, 289; purine bases in the

urine, 297, 298.
Kruger, T. R., 177.
Kru.mmacher, O., 70.
KuHN, J., 196.
KiTHNE, 459.

Kuhne, W., sugar, 92; digestion, 164.
KiJLZ, E., pentoses, 23; conjugated

glucuronic acids, 34; breaking-down of

sugar, 59, 71; glycogen, 68, 73, 75, 80,

92, 319; diabetes, 95; hydrcxy-butyric

acid, 97; cystine, 157.
KuLz, R., 410, 424.
KiJSTER, W., carbonic-oxide hemoglobin,

424, 561; hematin, 563, 564, 565;

bilirubin, 568.
Kuliabko, 90.
Kumagawa, M., .324, 632.
Kunkel, iron, 389, 395; arsenic, 407.
Kupffer, 411.
KuRAJEFF, D., 137, 208.
KuT-scHER, F., proteins, 135, 173, 176;

arginine, 156; digestion, 208.
Kyes, Preston, 115, 116.

Ladenburg, 154.
Lamy, 29.

Landergren, E., 345, 631, 467.
Landois, H., 125.
Landolt, H., 15.
Landsiedl, Anton, 200.
Landsteiner, K., 364. 688.
Landwehr, a. H., 4S.
Lang, S., 237.
Lange, Cornelia de, 369.
Langenbuch, 509.
Langendorff, 478.
Langendorff, O., 81.
Langebhans, 90.
Langstein, Leo, 160, 173, 271.
Langworth, C. F., 650.
Lapicque, L., 364, 389.
La Pl.vce, de, 334.
Laschkewitsch, 438.
Lassar-Cohn, 515.
Laurent, Em., 196.
Laves, E., 94.
Lavoisier, 70, 3.34.
Lawrow, .M., 208.
Lea, Sheridan, 71.
Le Bel, 15, 16, 25.
Leclerc du Sablon, 308.
Le Count, E. R., 115, 176.
Ledderhose, 35.
Lehmann, Curt, 394, 631.
Lehmann, K. B., 326.
Lehm.^^nn, K. H., 215.
Lemaire, F. a., 39.
Leo, H., 94.
Leone, S., 81.

Lepine, R., blood sugar, 32; glucolytic
ferment, 73, 87.

AUTHOR INDEX.

699

Leuchs, Hermann, glucosamine, 35;

amino-acids, 149, 151.
Levene, p. a., nucleic acid, 285; glu-

cothionic acid, 49; vagus nerve, 77;

gelatin, 176.
Le\v.\ndowski, N., 602.
Lbwinski, Johann, 556.
LiCKiERNiK, A., lecithine, 115; leucine, 148.

LlEBERMANN, 21.
LlEBBRMANN, LeO, 120.

LiEBiG, Justus v., 56, 69, 260, 277, 295,
356, 461.

LlEBREICH, 114, 613.
LiLIENFELD, L., 135.

LipPMANN, Ed.mund O. v., 13, 51, 90.

List, R., 123.

LoEB, J.vcQUEs, 358, 359, 672.

LoEB, Walther, 57, 70.

LoEBiscH, W. F., 266.

LoEW, O., 162, 450, 456.

LoEW, R. Mannan, 67.

LoEWi, Otto^ 213, 585.

LoEwy, A., cystinnria, 266; respiratory
exchange, 417, 422; blood circulation,
4.35; high altitudes, 436, 643.

LoRENz, N. v., 309.

Lubarsch, 123.

Lubarsch, O., 47.

LocA, S. DE, 490.

LUCHSINGER, B., 81.

LuciANi, L., 631.

LuDWiG, Carl, 70, 420, 486, 583.

LuDECKE, Karl, 113.

Luthje, H., 314, 320, 321.

LujcjANow, S. M., 352.

LuNiN, N., 355.

LusK, 82.

Lozzato, Riccardo, 23.

M.

Maar, v., 433, 434.

Macalldm, a., 389.

M.AC Calldm, 606.

Macpaydbn, a., 218.

MACLEOD, J. J. R., 233.

Magendie, 71.

Magnus, G., 409.

Magnus-Levy, Adolf, coma diabeticum,
98; Bence-Jones protein, 269 ; respiratory
exchange in diabetes, 323; formation of
carbohydrates from fat, 303; metabo-
lism, 630.

M.AIGNON, F., 97.

Malcolm, John, metabolism of salts, 403;

hypophysis, 610.
Malengreau, F., 1.39.
Man.asse, P.'.ul, 49.
Manche, Eduard, 68.
Mandel, John \., 49.
Mangold, Ernst, 507.
Maquenne, .306.
Marchal, E., 217.
M.vRCHLEwsKi, L., 396. 566, 567.
Marcuse, W., glycogen, 68; extirpation of

pancreas, 83, 87.

Mares, 290.

Marfori, Pig, 402.

Marggraf, 13.

Mark, H., 49.

Mark, W. R., 202.

Mar.x, 680.

Maschke, O., 123.

Massen, O., 231.

Mathews, A., 135, 140.

Matth.\ei, Gabrielle L. C, 51.

Mauthner, 215.

Maxwell, W., 42.

May, R., 632.

Mayer, A., 389.

Mayer, Hans, 388, 392.

Mayer, Robert, 334.

Mays, Karl, 168.

McChudden, F. H., 378.

Meinertz, 49.

Meisenheimer, J., 448, 465, 482.

Meissl, E., 309.

Mendel, Lafayette B., 296.

Merino, J. v., conjugated glucuronic acids,
34; diasta.se, 60, 71; absorption of
sugar, 61; phloridzin diabetes, 81, 82;
extirpation of pancreas, 82, 83, 86;
formation of glycogen, 92; gastric ac-
tivity, 507.

Mester, Bruno, 266.

Metzner, Rudolf, 587.

Meyer, H., 237.

Meyer, H.ans, glucuronic acid, 31 ; impor-
tance of fat, 112.

Meyer, Paul, 32.

Michaelis, 109.

MicHAELis, L., 670.

Michel, A., 124.

MiESCHER, Friedrich, metabolism of the
salmon, 130, 287, 351, 636; protein, 135;
nucleins, 140, 276, 284, 287; high
altitudes, 436.

Minkowski, extirpation of liver, 73; of
pancreas, 81, 82, 83, 86, 95, 321;
hydroxy-butyrie acid, 97; allantoine,
296; icterus, 570; uric acid, 237, 586.

MlTSCHERLICH, 461.

MiuRA, K., 95.

MiuRA, R., 632.

Moerner, C.\rl T., 49, 143.

MoERNER, K. .4. H., protein, 126, 143;

cystine, 157, 174, 176; pyroracemic acid,

169; (v-thiolactic acid, i69; acetanilide,

458; hemin, 565.
MoERS, 378.
MoHR, L., 290.

MoLiscH, Hans, 53, 125, 162.
Mombert, Paul, 645.
Montuori, a., 87.
Moore, 25.
Moos, 79.
MoRAWiTz, P., albumoses in blood, 211;

blood coagulation, 536, 541, 542, 547.
MoREAU, 4.3.3.
Morel, 387.
Morel, A., 548.

700

AUTHOR INDEX.

MoRGENROTH, J.', antifcnnents, 476;

hemolysis, 688. ,
MoniTz, 507.
MoRKowiN, N., 137.
MoRO, G4.

MOROCHOWETZ, LeO, 131.

Morris, 477.
Mo.sso, 547.
Muck, 378.

MOLLER, 117.

MiJLLER, Fr,\nz, iron, 389, .395; estimation
of oxygen, 413; higli altitudes, 430, 643;
estimation of blood, 557.

MtJLLER, FriedricH, glucosamine, 20,
142, 320; fasting experiments, 97, 394,
631 ; absorption of fat, 105; indole, 258;
intestinal putrefaction, 220; autolysis,
265, 478.

MuLLER, Johannes, 494.

MtJLLER, K.\RL, 41.

MiJLLER, Paul Th., 682.

MiJLLER V. Berneck, R., 468.

MuNZER, E., 230.

MuiRHEAD, Archibald, 232.

MuNK, Immanuel, fat absorption, 105,

106, 108; fat assimilation, 110, 309;

asparagine, 215; urea, 2.30; fasting

experiments, 394, 631; phenol, 458.
Musculus, M., conjugated glucuronic

acids, 34, 37; diastase, 59, 71.
Mylius, F., 515.

N.

Naegeli, C. v., 483.

Nagano, J., 62.

Nasse, Otto, 59, 71, 72, 73.

Naunyn, B., diabete.s, 77; glycocoll con-
jugate, 24.3, 458; icterus, 570.

Neesen, F., 413.

Neisser, M., 690.

Nencki, Marcel, physiological oxidation,
93, 440, 6.34; melanin, 145; digestion of
albumin, 152; glycocoll conjugate, 243,
244; albumin putrefaction, 153, 169;
intestinal bacteria, 218; urea, 228, 229,
230, 231; lactic acid, 378; behavior of
aromatic substances in the body, 458;
bile, 520;hematin, 396, 553, 557; gastric
juice, 462; urobilin, 572.

Nerking, Josef, 325.

Neubaner, Otto, 273.

Neuberg, Carl, pentoses, 22, 23, 24, 29;
glucuronic acid, 32, 33; chondroitin
sulphuric acid, 49, 143; cystine, 158,
266; behavior of stereoisomers in the
animal organism, 452.

Neumann, A., 281.

Neumeister, R., 152.

Nickles, J., 403.

NiCOLAIER, Artur, 586.

Niemann, A., 266.

Noeggerath, C. T., 355.

Noel-Paton, 71.

NoGUCHi, H., 115, 116, 690.

NoLF, P., formation of fibrinogen, 548;

biological reaction, 668.
Noll, A., 487.
Noorden, C. v., 290.
Nussbaum, Moritz, lungs, 429; kidneys,

583.
Nuttal, 670.
Nuttal, G. H. F., 64, 255.

O.
Obermayer, Friedrich, 192, 670.
Oddi, R., 49.
Oertmann, E... 409.
Orum, H. p. T., 514.
Ofner, Rudolf, 29.
Ogata, 104, 509.
Okunew, 208.
Oliver, G., 601.
Ollendorff, Gerhard, 58.
Oppenheimer, Carl, albumoses in blood,

211; ferments, 461, 481; biological

reaction, 670; toxines, 682.
Oppenheimer, Karl, 628.
Ord, William M., 605.
Orfila, M., 406.
Orgler, A., 49, 143.
Ortweileh, 219.
Osborne, T. B., nucleic acid, 23, 285;

protein, 139, 6.53.
Osborne, W. A., 359.
Osgood, R. B., 378.

OSTERTAG, R., 659'.
O.STWALD, 467.

o'sullivan, c, 467.

Oswald, Ad., 132, 607.

Otto, J. C, oxyhemoglobin, 125, 559;

methemoglobin, 562.
Overton, importance of fat, 112; effect

of ions, 358.

Pachon, v., 509.

Pages, 538.

Painter, C. F., 378.

Panceri, p., 490.

Panek, 269.

Parastschuk, S. W., 205, 505, 512.

Pasteur, 15, 470.

Patten, A. J., 159.

Paul, Theodor, 298, 593.

Pauly, Herm.^-NN, histidine, 154; adrena-
lin, 601.

Pavy, F. W., carbohydrates, 66, 71, 73;
phloridzin diabetes, 81 ; diabetes, 84.

Pawlow, J. P., 164, 478; rennin, 205, 206;
urea, 231; digestion, 485, 489, 496, 498,
499, 500, 501, 502, 505, 512, 520, 523;
relation of physiology to pathology, 668.

Payen, 39.

Pearson, 277.

Pekelharing, C. a., 462.

Peligot, 91.

Pellacini, p., 540.

Penzoldt, 494.

Perewoznikoff, 105.

AUTHOR INDEX.

701

Perls, 389.

Persoz, 39.

Pettenkofer, M.\x v., metabolism in
diabetes, 93; absorption of fat, 108;
assimilation of fat. 109; fat formation,
324; metabolism, 623.

Fetters, W., 97.

Pfeffer, W., 51.

Pfbiffer, R., 680.

Pfluger, Eduard F. W., carbohydrates,
6, 30, 44, 45, 46, 66, 78; physiological
combustion, 74, 459; glucosuria, 82, 83,
84, 85; absorption of fat, 104, 107;
formation of sugar, 314, 315, 316, 317,
318, 324, 329, 353 ; oxidation, 410; gases
of the saliva, 410; gases of the blood,
413, 415, 422, 423; source of muscular
power, 337; Alytes obstelricans (the
nurse-frog), 353.

PiccARD, J., 135, 277.

Pick, Ernst P., 192, 670.

PiLOTy, OsK.\R, 31, 32.

PiNKUs, S. N., 124.

PoDUSCHKA, Rudolf, 296.

PoHL, J., 230, 446.

POHL, Jdlius, 33.

POLITIS, 215.

Pollack, Leo, 168.

Popielski, L., 524, 528.

Porcher, Ch., 39.

Pouchet, G., 295.

Pregl, Fritz, egg-albumin, 172; carbon-
nitrogen quotient in urine, 270; albumin
derivatives in urine, 49, 270; bile acids,
515.

Preusse, C, 158, 251.

Preyer, W., 493.

Proscher, Friedrich, milk, 404; toad's
venom, 090.

Prout, William, 277.

Prutz, Wolfgang, 220.

Pry.m, Oskar, 531.

Qdincke, 389.

Q.

R.

Raaschoon, C. a., 284.
Raciborski, M., 446.
R.\dijewski, 105.
Radlkoper, L., 122.
Ramsden, W., 121.
. R.4NKE, J., 71.
Rapp, R., 464.
Raps, a., 413.
Raudnitz, p., 450.
Raudnitz, R. W., .392.
Rauschenbach, 540.
Re.^ch, Felix, 269, 312.
Reals, 83.

Rechenberg, v., 308.
Recoura, 333.
Reese, Heinrich, 264.
Regnadlt, v., 623.
Reichel, H., 467.

Reichert, B., 124.

Reinbold, Bela, trypsin digestion, 165,
167; edestiu, 172; nitric-o.xide hemo-
globin, 561.

Reiset, J., 623.

Renzi, db, 83.

Reverdin, Aug., 605.

Reverdin, Jacques Louis, 605

Rev, J. G., 373, 392.

Ribaut, H., 247.

RiBBERT, H., 582.

Richards, A. N., 411, 615.

Richardson, S. W. F., 617.

RiES, 255.

Run, J. J. L. v.\n, 21.

RiTTER, A., phloridzin diabetes, 81, S3;
uric acid, 594.

RiTTHAUSEN, H., 132, 169.

RocKWooD, Elbert W., 290.

RoEDER, Georg, 281.

RoHMANN, F., 62, 73, 102, 446, 595.

RoMER, Paul, 682.

RoHDE, Erwin, 152.

ROHRER, Ladisl.\us V., 492.

ROLLET, 550.

RoNA, Peter, histon, 174; breaking down
of polypeptides, 185, 192; albumin
synthesis in animal organisms, 213, 224,
355; Aspergillus niger, 216; proteolytic
ferments, 192, 506.

Rocs, E., 607.

Rosenfeld, Georg, fat, 110, 328; phlorid-
zin glucosuria (behavior of the liver),
322 ; estimation of fat, 325.

Rosenfeld, R., 30, 587.

Rcsenstein, Wilhelm, 81, 105, 108.

Rcstoski, Otto, protein, 134; gastric
digestion, 165, 192;edestin, 172;Bence-
Jones protein, 269; precipitin, 668, 682.

ROTHERA, C. H., 249.

RoTscHY, A., 563.

RoussiLLE, A., 308.

RuBNER, Max, utilization of fat, 108; for-
mation of fat, .309 ; ratio N:C in muscles,
324; isodynamics, 333, 335; metabolism,
628, 646, 655.

Rudolph, 398.

RuDziNSKi, Albin v. Rudno, 65.

Rudel, G., lime, 392; uric acid, 594.

Ruff, Otto, 58.

Sachs, Fritz, 288.

Sachs, Hans, 115, 688, 690.

Sachs, J., 51, .309.

Sahli, H., 543, 545.

Salaskin, S., erepsin, 167; leucinimide
169: plastein, 208; urea, 228; uric acid, 238!

Salkow.ski, E., pentoses, 21, 22, 323
glucuronic acid, 33; diastase, 71, 72
putrefaction of protein, 153, 169, 243
259; xantho-proteic reaction, 162, 163
urea, 228, 230, 2.34; sulphuric acid, 249,
uric acid, 290 ; allantoine, 295 ; adipocere,
326; antolysis, 478.

7U2

AUTHOR INDEX.

Salkowski, H., putrefaction of protein,
153, 169, 243.

Salomon, Georg, purine bases, 297,
298.

Salomon, H., 319, 434.

Salomon, Max, 90.

Salomon, W., 248.

Salvioli, Gaetano, 208.

Samuelt, Franz, melanin, 145; decom-
position of protein, 172; gliadin, 173;
breaking-down of polypeptides, 185,
228, 249, 452 ; building up and breaking
down of protein, 212, 245, 579, 639, 666.

Sandmeyer, W., 83, 95, 106.

Satta, Giuseppe, 98.

Sattler, Hubert, 389.

Sauer, 586.

Saussure, Theodore de, 51.

Sawit.sch, W., 522.

Sawjalow, 208.

Sch.Kfer, E. a., 601.

Schatternikoff, 440.

Scheele, Karl W., 277.

Scheermesser, W., 177.

ScHENK, Felix, 69.

Schenk, Fr., 73.

Schermetjewski, 94.

Schierbeck, N. p., 437.

ScHiFF, MoRiTZ, 77, 79, 606.

Schimper, a. F. W., 122.

Schindler, S., 285.

ScHiTTENHELM, ALFRED, casein, 174, 656;
elastin, 176; amino acids in urine, 264,
266, 452; cystinuria, 266, 267; nucleic
acid, 288, 289 ; breaking down of purine
bases, 290, 292 ; uricolytic ferment, 293,
294; uric acid, 10, 241; coagulation of
blood, 536.

Schlatter, C, 509.

schloesing, t., 196.

schlossberger, 402.

Schlossmann, Artur, 403.

Schmidt, Adolf, 582.

Schmidt, Alexander, hydrochloric acid
in the stomach, 203; oxidizable sul>
stances, 409; blood gases, 413; coagula-
tion of blood, 536, 537.

Schmidt, C, 42, 378.

Schmidt, Fr., 454.

ScHMIDT-MiJLHEIM, 546.

Schmiedeberg, O., hippuric acid, 9, 246,
247; glucuronic acid, 31; chondroitin
sulphuric acid, 49, 143; muscarin, 114;
proteins, 123, 135, 140; urea, 2.30, 234;
nucleic acid, 284, 285; oxydases, 440,
445; ferratin, 402.

Schneider, H., 447.

Schneidewind, 195.

schoffer, 420.

Schonbein, Christian P., 441, 461.

SCHONDORFF, Bernhard, 30, 47.

ScHOLz, W., 604.

Schottelius, 64.

schotten, g., 516.

Schreiber, 290.

ScHREUER, Max, 642.
ScHROEDER, W. V., Urea, 225, 231; uric
acid, 237, 241.

SCHROETER, F., 289.

ScHROETER, H. V., 435, 436.

ScHULTZE, Max, 411.

ScHULTZEN, O., diabetes, 93; urea, 228;
glycocoU conjugates, 243, 458; phos-
phorus poisoning, 255.

ScHULZ, F. N., galactosamine, 20; proteins,
122, 124, 127, 141; colloids, 128, 161;
cystine, 157; acid snails, 490; hemo-
globin, 558; metabolism, 633.

ScHULZE, B., 62, 309.

ScHULZE, E., cane sugar, 38; hemi-cellu-
lose, 42; lecithin, 115; amino acids, 155,
173; leucine, 148; phenylalanine, 151,
156; amides, 150, 151; ornithine, 155;
nitrogen cycle, 198; allantoine, 296.

ScHULZE, Ernst, 116.

Schu.mow-Simanowskaja, E. O., 498.

ScHUNCK, E., 567.

ScHUR, Heinrich, 287, 290.

Schwalbe, E., 5-36.

Schwarz, Hugo, 176.

ScHWARz, Leo, 99, 319.

Schwendener, 53.

Sczelkow, 70.

Seegen, J., diastase, 59, 72.

Seemann, J., 208, 574.

Seguin, 70.

Semon, 492.

Senator, H., 394, 631.

Senff, a., 81.

Senter, Georges, 450.

Setschenow, 421, 423.

Shaffer, Philipp, 450.

Shedd, 446.

SiAU, R. L., 73.

SiEBER, N., physiological oxidation, 93,
440, 634; melanin, 145; intestinal
bacteria, 218; significance of hydro-
chloric acid in the stomach, 219; lactic
acid, 378; gastric juice, 462; hematin,
563 ; urobilin, 572.

Siegfried, M., jecorin, 49; kyrin, 177;
carbamic acids, 233, 242, 425.

Sigmund, W., 103.

Simon, Oskar, 318.

S.IOQVIST, John, 120.

Skita, Aladar, 176.

Skraup, Zd. H., 177.

Slowtzow, B., btoffwechsel pentosans, 65;
metabolism, 628.

Smith, J. Lorrain, 431.

SOBIERANSKI, W. V., 582.

SociN, C. A., diabetes, 95; iron, 355.
Soldner, 368.
S<S?RENSEN, S. P. L., 152.
Sollmann, Torald, 584.
Sommer, a., 328.
Sonden, Klas, 70, 623, 631, 647.
SOXHLET, F., 309.
Spallanzani, 203, 219, 461.
Speck, C, 70.

AUTHOR INDEX.

703

Spiess, a., 486.

Spiro, K., 467, 547.

Spiro, p., 75.

Spitzer, W., uric acid, 10, 289 ; oxydases,
446.

Stadelmann, E., 152, 571.

Stadelmann, Ernst, 99.

St.^dth.^qen, 266.

St.^helin, Rudolf, 643.

Stahel, H., 543.

Starling, E. H., secretin, 168, 527; inner-
vation of the intestine, 507 ; lymph, 574.

Stass, J. S., 30.

Steiger, E., 42, 155.

Stein, Gustav, 117.

Steinfeld, Wladimir, 392.

Stern, Hans, 570.

Stern, L., 449.

Sternberg, Wilhelm, 494.

Steudel, H., glucosamine, 35, 143; uracil,
281 1 thymine, 281 ^ cytosine, 281 ; nucleic
acid, 285 ; behavior of pyrimidiue bases
in the organism, 298.

Stohmann, F., 62, 333.

Stoklasa, Julius, 73.

Stone, W. E., 64.

Strassburg, G., 435.

Straub, Walter, 81.

Strauss, H., 29.

Strecker, Adolf, lecithine, 1 12 ; creatine,
235; uric acid,259,302;bileacids,514,515.

Strohmer, F., 309.

Suter, 169.

Suzuki, Umetaro, cystine, 159, 266,
267, 291.

SwiRSKi, G., 389.

Tait, 615.

Takaki, T., 687.

Takamine, J., 601.

Tallquist, F. W., 344.

Tammann, G., 403.

Tangl, Franz, 73, 655.

Tappeiner, H., 62, 63.

Tarcha.vopp, Johannes Furst, 570.

Tartakowsky, 389.

Tauber, 253.

Taylor, 328.

Tchistowitsch, 668.

Tebb, M. C, 60.

Teichmann, M., 107.

Tengstrom, Stephan, 515.

Teruuchi, Yutaka, protein, 173; decom-
position of polypeptides, 185, 192, 228.

Tb.sti, 39.

Thanhofper, L. v., 106.

Thenard, grape-.sugar, 90; H,0,, 450.

Thiel, Andreas, 82.

Thiele, O., 270.

Thierpelder, H., cerebron, 20, 613;
glucuronic acid, 31, 32, .34; life without
bacteria, 64, 255' fermentation of
sugars, 471.

Thiroloix, J., 83.

Thompson, W. H., 136; urea, 228;

hypophysis, 610.
Thudichum, J. LuDWiG W., 614.
Tichomiroff, a., 287.
Tigerstedt, Robert, metabolism, 70,

623, 631, 6.32, 647.
Tobler, LuDwiG, 507.
ToLLENS, B., 13, 289.
ToMPSON, 467.
ToRUP, 422.
Traube, J., 532.
Traube, Moritz, oxidation, 444, 445;

H.O,, 449.
Trebou.x, 57.
Troschel, 490.
Tscherwinsky, N., 310.
Tsuji, 66.

U.
Udranskt, L. v., 266.
Uhlbnhut, 670.
Underhill, Frank P., 587

V.
Vallisneri, 39.
Vass.\le, 606.
Vaudin, L., 373.
v.\uquelin, 277.
Vernon, H. M., erepsin, 168; heat rigor,

617.
Verwor.n, Max, 459.
ViELLE, 333.
ViRCHOw, Rudolf, 569.
ViTEK, Eugen, 73.
ViJLTz, W., 215.
Vogel, J., 196.
VoGEL, J., 23, 59, 71.
VoisiN, S., 273.
VoiT, Carl, glycogen, 66; muscular power,

69, 70; consumption of material in

diabetes, 93 ; absorption of fat, 108, 309 ;

asparagin, 215; metabolism, 343, 352,

623, 631, 6.32, 635, 636; lime, 375;

formation of fat, 324.
VoiT, Erwin, glycogen, 66; estimation of

fat, 325; adipocere, 326; lime, 375;

metabolism. 6.33.
VoiT, Fritz, sugar, 39, 61, 323; galactose

in diabetes, 95.
VOLHARD, 104.

Volhard, J., 2.35.
VooRNVELD, H. J. A. van, 4.36.
Vossius, Adolf, 570.

VULPLYN, 601.

w.

Wahlgren, v., 514.
Waldvogel, 290.

Walter, Friedrich, 100, 230, 241.
Walter, G., .388.
Walther, a., 499, 525.
Warburg, Otto, 475, 494.
Warren, J. W., 75.
Wartenburg, H., 103.
Wassermann, 680, 687.

704

AUTHOR INDEX.

Webeh, S., 631.

Wechsberg, F., 690.

Weigert, Fritz, 155.

Wbinland, Ernst, invertin, 61; lactose,

62, 323; antiferraents, 477.
Weinmann, R., 494.
Weintraud, W., 94, 98, 299.
Weiske, a., cellulose, 02, 64; asparagin,

216; fonnation of fat, 309.
Weiss, S., 68.
Wells, H. G., 176.
Wenzel, Friedrich, 30.
Werner, A., 15.
Werther, Moritz, 75.
Wheeler, Henry L., 282.
White, Benjamin, 296.
WiECHOwsKi, Wilhelm, 242, 24.5, 246.

WiEDERSHEIM, R., 100.

Wiener, Hugo, 10, 238, 290, 294, 299.
Wild, E., 309.
Wild, W., 449.
Will, A., 105.

WiLFARTH, H., 196.

WiLLi.Ans, Francis, 388.

WiLLSTADTER, 113.

Windaus, a., cholesterol, 117; histidine,

154; imidazole, 286.
WiNDi.scH, Karl, 326.
WiNOGRADSKY, S., 193, 194, 195.
Winter, C, 470.
Winternitz, R., 4.38.
Winterstein, E., tunicin, 42; protein

cleavage products, 154, 173; ornithine,

155; arginine, 156.

WiSLICENUS, 515.

Wislicenus, J., 69, 412.

Wittich, v., 71.

Witzel, O., 83.

WoHLER, F., hippuric acid, 5, 50, 242,

243, 479; uric acid, 236, 277; allantoine,

295; emulsin, 461.

WciRNER, Emil, 613.

WoHL, A., 58.

Wohlge.mdth, J., pentoses in organs, 22;

cystine, 249; behavior of stereoisomers

in the organism, 452.
Wolff, E., 62.
Wolff, Gustav, 674.
Wolpfberg, Siegfried, 429.
WoLKOw, M., 270, 271.
wollaston, 157.
Woltering, W. F. C, 389, 401.
worm-muller, 95.
Wortmann, J., 63.
Wroblewski, 654.
WURTZ, A., 113.

Y.
Young, William J., 47.

Z.

Zaleski, J., urea, 231; hematin, 396, 563,
567; urobihn, 572.

Z.ANETTI, C. N., 161.

Zawarkin, 106.

Zerner, Theodor J., 594.

Zeyneck, Richard v., 562.

Ziegler, E., 458.

Zillesen, 75.

ZlNOFFSKY, O., 125, 384.

Zinnser, Adolf, 104.

Zschokke, 664.

ZsiGMONDY, R., 128, 161, 361.

Zuntz, N., cellulose, 62; muscular power,
70 ; phloridzin diabetes, 82 ; gas exchange,
420, 428; blood gases, 422, 424, 426;
high altitudes, 436, 643; fasting experi-
ments, 394, 631.

Zwerger, R., 177.

SUBJECT INDEX.

Abrin, 681.

Abrus prjEcatorius, 681.

Absorption of carbohydrates, 65; of fat,
107; of lime, 373; of iron, 388; of
gases, 413; in the stomach, 508; in
the intestine, 532.

Absorption coefficients (of the blood for
O, N and CO2), 415; (of serum for
CO,), 421.

Absorption spectrum of oxyhemoglobin
and its derivatives, 560 et seq.

Acceptors. See Side-chain theory.

Acetal, 303.

Acetanilid, 458.

Acetic acid, formation from carliohy-
drates in the inte.stine, 63; in the
tissues, 74; f rom glycocoll, 170; from
bacterial action, 465; as product of
alcoholic fermentation, 483.

Acetic acid bacteria, 448.

Aceto-acetic acid, 97.

Acetone, a cause of glucosuria, 81; elimi-
nation, 97; from leucine, 454; in
preserved yeasts, 464.

Acetonuria, 97, 98.

Acetone bodies, source of, 97, 98, 99, 454.

p-Acetyl-amido-phenol, 45S.

Achras sapota, 39.

Acid albumins, 121, 203.

Acidity of blood, 99; of urine, 591.

Acidosis, 99, 242.

Acid, chemical and physico-chemical con-
ceptions, 591, 592; secretion of by
snails, 490.

Acid fuchsin, 583.

Acipenser stellatus, 137.

Acipenserin, 137.

Acromegaly, 609.

Activation of muscle ferment, 88.

Activity, optical, 15.
hypertrophic, 350.

Acrose, 15.

Adaptability, of salivary secretion, 489;
of gastric secretion, 501.

Adaptation, chromatic, 160.

Addison's disease, 603.

Adelomorphous cells, 496.

Adenine, 280, 297.

Adhesion, importance in blood coagula-
tion, 545.

Adipocere, 326.

Adiposity, 111.

Adonis vemalis, 29.

Adonitol, 29.

Adrenalin, 601 et seq.

Aerobic bacteria, 412.

Agar-agar, 42.

Age, influence on metabolism, 629.

Agglutination, 680.

Alanine, 148, 304; action on muscles, 358;
taste, 490.

Alanine anhydride, 228 et seq., 180, 186.

Alanine carbonate of calcium, 233.

Alanyl-alanine, 179, 180, 185, 228, 474.

Alanyl-glycine, 179, 180, 186, 474.

Alanyl-glycine anhydride, 186.

Alanyl-glycyl-glycine, 474.

Alanyl-leucine, 179, 18t, 474.

Alanyl-leucyl-glycine, 474.

Albumin. See also Protein.

Albumin, absorption of, 208; denatur-
ization of, 120, 121; from carbohy-
drates, .304; calorific value of, 3.33-
336; with regard to isodynamics,
336; as source of muscular energy,
337; as source of energy for gland-
work, 341; influence upon general
metabolism, 347, 348; circulating
and organized, 673 et seq.; content
of foodstuffs, 648.

Albumin bodies, substituted, 127; clas-
sification of, 129 et seq.; size of
molecules, 127, 128; simple, 131;
compound, 131; true, 131.

Albumin crystals, 121, 122, 125; fatten-
ing, 618, 642.

Albumin decomposition, in Coma dia-
beticum, 100; by ferments, 166 et
seq.; in phosphorus poisoning, 328;
computation of extent of, 622, 627.

Albumin derivatives in urine, 270,271;
requirement, 221 et seq., 342, 343,
639 et seq.; with carbohydrates or
fats, 342 et seq.; with mixed diet,
342, 343.

Albumin metabolism, 6^, 637.

Albumin synthesis, in the intestine, 208;
in animal body, 213; by fungi, 216;
in plants, 193, 201 et seq.

Albumin putrefaction, 169, 170, 217, 218,
253.

Albuminous glands, 485.

Albumins, 131, 172. See also the vari-
ous albumins.

Albuminates, 422.

Abuminoids, 131, 176.

Albumoids, 138.

706

SUBJECT INDEX.

Albuminous bodies. See Proteins.

Albuminuria, 268.

Albumoses, 164.

Aleapton, 270.

Alcaptonuria, 270.

Alcohol, formation from carbohydrates in
the intestines, 63; influence upon
gastric secretion, 500; upon pan-
creatic secretion, 527.

Alcoholic fermentation, 27, 461, 469, 482,
48.3.

Alcohohc poisoning, 327.

Alcohols, in the tissues, 74 ; of sugars, 27.

Aldoses, 19.

Aleuron grains, 122.

Algae, fresh water, 52 ; salt water, 52.

Alimentary glucosuria, 30, 76.

Aliphatic amino acids, 147.

Alizarin, 21.

Alkali albuminates, 121.

Alkali salts, part played in CO, combina-
tion, 421-424 ; migration in the blood,
426.

Alkalinity, influence upon oxidation, 440.

Alkalinity of blood, 421.

Alkaloids, 201, 305.

Allantoine, 277, 295.

Allantoic fluid, 277.

Alloxan, 277, 279.

AUoxuric bases, 297.

AUoxyproteie acid, 270.

Almonds, 133, 308, 381.

Alveolar air, composition, 428, 429; gas
tension, 414; carbon dioxide ten-
sion, 429.

Alj-tes obstetricans, 353.

Amanita muscaria, 114.

Amanitine, 114.

Amaryllidace:e, 45.

Amboceptor, 689.

Amides, 150.

p-Amido phenol, 252, 458.

Amino acids, 129, 146 et seq. ; cleavage
of inactive acids into optically active
components, ISl ; part assumed in
CO2 union, 233, 425; transformation
into sugars, 317 el seq.

Amino acid chloride, 180.

Amino-acetic acid. See Glycocoll.

Amino-benzoic acids, 234.

Amino-butyric acid, 148.

Amino-butyryl-amino-butyric acid A and
B, 474.

Amino-butyryl-glycine, 474.

Amino-caproic acid. See Leucine.

Amino-ethyl sulphonic acid. See Taurine.

Amino-glutaric acid. See Glutamic acid.

Amino-^-hydroxypropionic acid. See
Serine.

Amino-hydroxy-valeric acid, 152.

Amino-/3-imida2ol-propionic acid. See
Histidine.

Amino-isobutyl-acetic acid. See Leucine.

Amino-isovaleric acid, 148, 454, 494.

Amino-isovaleryl-glycine, 474.

Animo-niethyl-ethyl-propionic acid. See

Isoleucine.
Amino-, 6-, oxypurine, 2-. See Guanine.
Amino-, 2-, oxypyrimidine, 6-. See Cy-

tosine.
Amino propionic acid. See Alanine.
Amino pyrotartaric acid. See Glutamic

acid.
Amino purine, 6-. See Adenine.
Amino salicylic acids, 0- and p-, 234.
Amino-succinic acid. See Aspartic acid.
Amino-thiolactic acid. See Cystine.
Amino-valeric acids, 148, 170, 454, 494.
Amino sugar. See Glucosamine.
Ammonia, 99, 146, 193, 231, 237.
Ammonium carbonate, 229, 231.

chloride, action upon blood corpuscles,
550.

cyanate, 231.

formate, 231.

lactate, 238.

oxalate, 217.
Amniotic fluid, 236.
Amceba, 47, 4.59.
Amorphophallus Konjako, 29.
Amphibia, cutaneous respiration of, 432.
Amygdalin, 20, 480.
Amyl alcohol, 148, 470.
Amylan, 44.

Amyl nitrite, cause of glucosuria, 81.
Amylodextrin, 43.
Amyloid, 49, 1.38.
Amylolytic ferment, 59, 60.
Amylum, 42.
Ansmia, 386.
Anaerobic bacteria, 412.
Anchylostoraum caninum, 547.
Animal foods, value of, 650 et seq.
Anions, 358 et seq.
Amielida, 360.
Anodons, 567.
Anthrax bacillus, 153.
Anti-catalyzers, 468.
Anti-ferments, 476.
Anti-rennin, 476.
Antimony poisoning, 327.
Antitoxins, 476, 684 et seq.
Antodon rosacea, 672.
Antoxyproteic acid, 270.
Apiin, 36.
Apiose, 36.

Appetite, effect on gastric secretion, 498.
Araban, 65.

Arabinose, 23, 24, 44, 452.
Arabite, 28.
Arabonic acid, 28.
Arachidic acid, 102.
Arachnolysin, 690.
Arbacia pustulosa, 140.
Arginase, 228.
Arginine, 154, 226, 259.
Arginyl-arginine, 179.
Argon, 414.

Aromatic amino acids, 151.
Arsenic, 81, 406.

SUBJECT INDEX.

707

Arsenious acid, 446.
Arsenic acid, 446.
Arsenic poisonin", 327.
Arseniureted hydrogen, 570.
Arsine. See Arseniureted hydrogen.
Ash of milk, 395.
of sucklings, 367, 368.
of foods, 369.
Asparagine, 150, 202, 215, 358.
Asparagus, asparagine, 150; iron, 381.
Asparagyl-dialanine, 182.
Asparagyl-monoglycine, 182.
Aspartic acid, 149, 304, 470.
Aspergillus oryzae, 37.
Aspergillus niger, 217.
Assimilation of fats, 105; of iron, .392; of

lime, 466 et seq.; of sugar (direct),

55; of carbonic acid, 51.
Assimilation limit for carbohydrates, 76,

88.
Assimilation product of CO,, 54.
Ass's milk, 404, 655.
Asymmetric carbon, 16.
Asymmetric cleavage of polypeptides,

184, 185; of racemic amino acids,

228, 443.
Asymmetric synthesis, 54 et seq.
Atractylis, 29.
Atriplex, 362.
Autolysis, 265, 266, 479.

Bacillus Dellbriicki (Leichmann), 465.

B.acfiUus putrificus, 218.

Bacteria, as reagent for o.xygen, 53.

aerobic and anaerobic, 412.
Bacterial action upon nucleins, 289.
Bacterial poisons, 680 et seq.
Bacterium coli, 153, 218.
Bacterium denitrificans, 195.
Bacterium lactis, 27.
Bacterium lactis aerogenes, 218.
Bacterium radicicola, 197.
Bacterium thermo, 63.
Bacterium xylinum, 448.
Barley, gliadiu, 133; iron, 380.
Bathing sponge. See Sponge.
Balloon ascension, 436.
Beans, iron content of, 380.
Beaker cells. See Goblet cells.
Beef, 366, 370, 377, 381 ; ash of, 369, 370,

371 ; extract of, 500.
Beef-blood, 366.
Beer-acetic-acid-bacteria, 465.
Beeswax, 102.
Beggiatoa, 194.
Bence-Jones protein, 269.
Benzaldehyde, 20.
Benzamide, 244.
Benzene, 93, 252. '►

Benzidine, 446.
Benzoic acid, 5, 229, 440, 446.
Benzyl alcohol, 440, 446.
Bertholletia, 123.
Betain, 114.

Betulase, 20.

Bile, 513; influence on digestion of fats,
104, 107, 519 ; antiseptic action, 220,
519; composition, 516, 517; influ-
ence on peristalsis, 519; influence on
lipase, 520.

Bile-acids, 514 e( seq.

Bile concretions, 572.

Bile pigments, 513, 569 et seq.

Bilianic acid, 515.

Biliary fistula, 520.

Bilirubin, 569.

Biliverdin, 569.

Biological reaction, 668 et seq.

Birch, cane-sugar in, 38.

Birds, pancreas extirpation, 583; dia-
betic puncture, 77; oil bags of, 102,
595

Bismuth, 392.

Biuret base (Ciirtius'), 474.

Biuret reaction, 163.

Bladder-stones, 297.

Blood, sugar in, 29, 30; jecorin, 49; glu-
colytic ferment, 73; fat, 109; urea,
225; creatine, 235; impoverished,
386; gases of, 413 et seq.; defibri-
nated and clotted, 536; analysis of,
553 et seq. ; amount of, 556.

Blood clot, 535.

Blood coagulation, 535 et seq.

Blood-corpuscles, possible presence of his-
ton, 135; presence of nucleo-proteid,
140, 141; formation of, 401; union
with CO,, 423 et seq. ; behavior in high
altitudes, 436; red and white, 5.35;
red, 549, 550, 556; white, 551, 556.

Blood e.xtravasations, 569.

Blood gases, 408 et seq.

Blood, laked, 550.

Blood-leech. See Leech.

Blood-pigments, 124.

Blood pressure, effect on secretion of
urine, 584; influenced by adrenalin,
600 et seq.

Blood plates, 535, 552.

Blood-vessels, glycogen content of, 47.

Blood relationship, 670 et seq.

Boar, spermatozoa from, 285.

Bog iron ore deposits, 193.

Bones, mucoid in, 143; in rachitis, 371;
in osteomalacia, 377; marrow of,
395, 573; influence of castration, 600;
influence of thyroid gland, 605; tissue
of, 611.

Bottcher's sugar test, 25.

Bowman-Miiller's capsule, 581.

Boyle's law, 413.

Brain, glycogen in, 47; phosphorus, 403;
creatine, 236.

Brain substance, affinity to tetanus toxin,
687.

Bread, iron and lime content, 370, 377,
380; influence on gastric secretion,
502, 503; upon pancreatic secretion,
531.

708

SUBJECT INDEX.

Brom-capronyl-glycine-chloride, 181.
Brom-iso-capronyl-chloride, -, 181.
Bromine water, reagent for tryptophane,

152.
Brom-phenyl-mercapturic acid, 158.
Brom-propionyl chloride, ISO.
Brunner's glands, 512.
Buchu seeds, 114.
Bull, .spermatozoa of, 285; blood of, 554,

555.
BurssB mucosae, 578.
Butter-fat, 333.
Butyric acid, 102 ; from carbohydrates in

the intestine, 63, 04.
Butyric acid fermentation, 27, 469.
Butylchloral hydrate, 34.

C.

Cabbage, iron and calcium content, 370,
381.

Cachexia strumipriva, 605.

Cadaverine, 154, 170, 259.

Caffeine, 200, 280.

Caecum, elimination of iron, etc., 389
et seq.

Calcium, 80, 370 et seq.

Calcium chloride, antidote for NaCl
glucosuria, 80 ; effect on blood coagu-
lation, 538.

Calcium salts, in foods, 370-372; in
rachitis, 371; elimination of, 392;
influence on blood coagulation, 538.

Calories, definition of, 332, 333.

Calorific requirement, 645-647.

Calorific value of certain foods, 333, 334,
336, 661, 662.

Camphene glycol, 34.

Camphor, 32, 34.

Cane-sugar, 13, 29, 37, 190; inversion in
the intestine, 61; behavior when
introduced directly into the blood,
61 ; effect upon the muscles, 323,
358; calorific value, 333, 336.

Caoutchouc, 339, 312. .

Capric acid, 102.

Caproic acid, 102.

Caprylic acid, 102.

Caramel, 25.

Carbamate of ammonium, 230.

Carbamic acids, 232, 425.

Carbaminobenzoic acid, 234.

Carbamic acid amide, 232.

Carbaminoisethionic acid, 234.

Carbamo-acetate of calcium, 233.

Carbamo-propionate of calcium, 233.

Carbohemoglobin, 562.

Carbohydrates, 13-100; transformation
into fat in plants, 103; as source of
heat, 342 ; influence upon the protein
requirement, 342 et seq.; influence
upon fattening, 345 et seq.; influence
on respiratory quotient, 346; influ-
ence on general metabolism, 347

Carbohydrate group of the proteins, 141,
159, 317.

Carbohydrate metabolism, 623, 627, 632.

Carbon atom, asymmetric, 16.

Carbon atom added to sugars, 18.

Carbon chains of amino acid radicles, 318,
324.

Carbon, relation to nitrogen in urine, 321.

Carbon dio.xide (Carbonic acid), assimi-
lation of, 15, 51; formation in the
intestines from carbohydrates, 63;
gas exchange, 420; gas pressure in
blood, 420 et seq.; in lymph, 435;
absorption in serum, 421 ; influence
on composition of blood components,
426; influence on absorption of
oxygen, 427.

Carbon monoxide glucosuria, 81.

Carbon-monoxide hemoglobin, 424, 561.

Carcinoma, 675.

Carica papaya, 168.

Caries of the teeth, 493.

Carmine, elimination by the kidneys, 583.

Carnine, 660.

Carrots, iron content, 381.

Carp, eggs of (hematogen), 388; sperma
of, 136; spermatozoa, 285.

Cartilage, chondroitin-sulphuric acid, 49;
mucoid, 143; tissue, 611.

Casein, 134, 206; from cow's milk 174;
from goat's milk, 174; calorific
value, 333; glutamic acid content,
653.

Castor-oil seeds, 103, 122.

Castration 600; influence on osteoma-
lacia, 378.

Catalases, 449.

Catalyzers, 467.

Catalysis, 467.

Catechol, 254.

Catechoyl sulphuric acid, 251, 254.

Cations, 358 et seq.

Cats, rate of growth, 371, 404; blood,
554, 555; hemoglobin, 125, 559.

Cell metabolism, 311 et seq., 349 et seq.

Cell nucleus, 672.

Cellobiose, 37.

Cellose, 38.

Cells, chief or principal, 495; composition
of, 329, 330.

Cell-substance, resistance towards fer-
ments, 191.

Cellulase, 482.

Cellulose, 13, 31, 38, 41, 42, 62, 63, 482.

Celtis reticulosa Miq., 201.

Cement, of tooth, 493.

Cephalopoda, 402, 447.

Cerealose, 39.

Cerebrin, 613.

Cerebron, 20, 31, 613.

Cerebronic acid, 20, 31, 613.

Cerebro-spinal fluid, 615.

Cerosin, 44.

Cetin, 102.

Cetyl alcohol, 102.

SUBJECT INDEX.

709

Chemical energy, 52.

Chenopodium, 302.

Cherries, iron and lime in, 64.

Cherry-gum, 24, 42.

Chief cells, 495.

Chilodon, 46.

Chitin, 162.

Chiton, 402.

Chloracetyl chloride, ISO.

Chloracetyl-glycine, ISO.

Chloral, 81.

Chloral hydrate, 32, 34.

Chloride of sodium, 361 et seq.

Chlorine, 435.

Chlorine water reagent for tryptophane,

152.
Chloroform, 81, 327.
Chlorophyll, 52, 396, 397, 565, 566.
Chlorosis, 386 et seq.
Cholalic acid, 229, 248, 514.
Choleic acid, 514.
Cholera bacteria, 679.
Cholesterol, 116, 117, 514, 613.
Cholic acid. See Cholalic acid.
Choline, 112, 113,615.
Chondro-albumoid, 138.
Chondroitin, 143.

Chondroitin-sulphuric acid, 49, 138.
Chondromucoid, 143.
Chorda dorsalis, 138.
, Chorda tympani, 485.
Chroraogens in suprarenal capsule, 601.
Chromophyll, 52, 565.
Chyle, 107.
Chyme, 506.
Cilianic acid, 515.
Circulation, of foetus, 410.
Cladophora, 357.

Classification of proteins, 129 et seq.
Cleavage. See Hydrolysis.
Cleavage processes, as source of energy,

74, 411.
Cleavage products of proteins, 146-170.
Clostridium Pasteurianum, 195.
Clupeine, 136, 175.
Coagulation of proteins, 120, 121 ; of

fibrinogin, 133; of blood, 535-544.
Cocoa beans, iron and lime content, 370,

380.
Cobitis fossilis, 438,
Cobra poison, 115, 117, 547.
Cocks, castration of, 600.
Coefficient of absoqjtion. See Absorption.
Colloid, 608.
Colloids, 120, 360.
Colorability of tissues, 678.
Coloring matters. See various pigments.
Colostrum, 655.
Coma diabeticum, 99.
Combustion, in the organism, 409 et seq.,

439-460.
Comparative chemical investigation, 2,

663 et seq.
Complement, 689.
\Composition of body cells, 329, 3.30.

Concept, of quantity, 1 1 ; of food, 349-354.

Conchiolin, 138.

Configuration, of he.xoses, 17, 18; influ-
ence on fermentation, 470 et seq.;
on taste, 494.

Conglutin, 173; calorific value, 333; glu-
tamic acid content, 653.

Connective tissue, 47, 611.

Coniferise, 199.

Constitution of proteins, 171, 178.

Consumption of foodstuffs, 647.

Contact action, 467.

Control experiment, 8.

Coprosterol, 117.

Coregonus oxyrhynchus, 137.

Cornea, 143.

Copper albuminate, 128.

Copper in hemocyanin, 402.

Copper salts, 357.

Corn, zein from, 133.

Corpora amylacea, 138.

Cotton-seed, albumin crystals, 123.

Cow's blood, 554, 555; hemoglobin from,
559

Cow's milk, 366-380, 404.

Crab's muscle, 546, 576.

Creatine, 235.

Creatonine, 235.

Cresol, P-, 170, 252, 255.

Cresol-sulphuric acid, p-, 254.

Cretins, 604.

Crustaceans, 402.

Crystalline lens. See Lens.

Crystallization of proteins, 122.

Cucurbita, 103.

Cupric oxide, as carrier of oxygen, 445.

Curari, 30, 81.

Cyanamide, 156, 235.

Cyanhydrine, 18.

Cyanic acid, 230, 278.

Cyanophyceae, 125.

Cyanuric acid, 238.

Cycadeae, 198.

Cyclopterine, 137, 175.

C^cloptenis lumpus, 137.

Cycle, of carbon, 51, 200; of hydrogen, 53,
200; of oxygen, 51; of nitrogen, 194;
of sulphur, 200.

Cyprinines, 137, 175.

Cyprinus carpio, 137.

Cysteine, 158.

Cysteinic acid, 158.

Cystine, 157, 239, 245, 248, 266, 304.

Cystine diathesis, 267.

Cystinuria, 260, 266.

Cysts, 142.

Cytosine, 282, 298.

D.

Dahlias, inulin content, 29.
Dalton's law, 413.

Dandelion greens, iron content of, 381.
Dates, iron and lime content, 370, 380.
Death rigor, 133, 617, 618.

710

SUBJECT INDEX.

Decomposition of polypeptids in the
organism, 185; of proteins by tryp-
sin, 188, 189, 208; by pepsins, 203;
of the purine bases, 292; of the
pyrimidinebases, 298; of albumin, 193,
260 ; of albumin in intestines, 209, 212 ;
in germinating seeds, 201, 202.

Degeneration, fatty, 327.

Dehydrocholic acid, 515.

Deilephilia elpenor and euphorbia?, 447.

Delomorphous or parietal cells, 496.

Denaturizing of proteins, 120.

Denitrification, 194.

Denitrifying bacteria, 198.

Dentin, 493.

Desamidation, 232, 304.

Development, rate of (relation to com-
position of milk), 367, 370, 371, 404,
655.

Dextrins, 43, 44, 46, 59, 61.

Dextrine-like substances in urine, 48, 96.

Dextrose, 29. See (/-Glucose.

Diabetes mellitus, 87 el seq., 332, 341.

Diabetic puncture, 77.

Dialanyl-cystine, 179, 185, 474.

Dialuric acid, 240.

Dialysis, 120.

Diamino acids, 129.

Diamino-capronic acid a and e, 154.

Diamino- /3-dithiodilactyl acid, a, 1.58.

Diamino-mono carboxylic acids, 1.54.

Diamino hydroxy-mono-carboxylic acids,
154.

Diamino-trihydroxy-dodecanoic acid, 154,
318.

Diamino-valeric acids, 156.

Diastase, 39, 40, 476; of saliva, 59, 490;
of pancreatic juice, 60, 521 ; of plants,
477; of intestinal juice, 512.

Dibenzoyl-ornithine, 155.

Dicalcium phosphate, 122.

Diet for various classes of people, 644 et seq.

Digestibility, 507.

Digestion, 59-62, 104-110, 203-220, 579,
638.

Digitalin, 21.

Digitonin, 21, 31.

Digitoxin, 21.

Diglycyl-glycine, 180-228.

Dihydrocholesterol, 117.

Diketopiperazine, 180.

Dileucyl-cystine, 179, 185, 474.

Dileucyl-glycyl-glycine, 179, 217, 475.

Diraethyl-amino-benzaldehyde, p-, 152.

Dimethyl-cyclo-octadi-ine (15), 306.

Dimethyl-dihydroxy-purines, 280.

Dimethyl-xanthine, 297.

Disodium urate, 299.

Diontea museipala, 168.

Dihydroxybenzenes, 252.

Dihydroxyphenylacetic acid, 257, 271.

Dioses, 19.

Dioxypurine, 280.

Dioxypyrimidine, 281.

Dipaimitylolein, 102.

Diphtheria toxin, 680.

Disaccharides, 36-40.

Disinfection by HCl of stomach, 218, 219.

Dissociation, of hemoglobin, 416; of bicar-
bonate 422.

Distearylpalmitin, 102.

Dog, ash of suckling, 367; rate of develop-
ment, 404.

Dog's blood, oxygen content, 415; COj
content, 421; composition, 554-555;
hemoglobin, 559.

Dog's milk, composition, 366, 404.

Dolium galea, 490.

Doris, 402.

Drosera, 168.

Drosophila funebris, 448.

Dulcite, 27.

Duodenum, absorption of iron, 388 et seq.;
digestion in, 511 et .^eq.

E.

Earth-worms, glycogen in, 46.

Echinoderms, 46, 493.

Echinus eggs, 672.

Eck's fistula, 231.

Edestin, 126, 127, 132, 172.

Edinger's theory of nervous exhaustion,
616.

Eggs, composition of, 659.

Egg albumin, 124, 132, 172, 333, 653.

Egg globulin, 132.

Egg-yolk, 370, 377, 381, 3S7.

Eteagnacffi, 197, 198.

Elastin, 138, 176.

Elimination of iron, 388; of metabolic
end products, 579-595.

Elodea canadensis, 57.

Emulsin, 20, 461, 472.

Emulsions, 103, 105.

Enamel, .527.

Endotryptase, 464.

End-products of protein metabolism, 221-
250.

Energy, chemical, 52; obtained by
cleavage, 74, 412, 441; obtained by
oxidation, 74, 436—460; from food,
331, 332; radiant, 52; conservation
of, 334; exchange of in animals of
different sizes, 629.

Enterokinase, 208, 521.

Enzymes. .See Ferments.

Epiguanine, 298.

Episarkine, 298.

Epithelium, 47.

Erepsin, 167, 513, 531.

Erucic acid, 110.

Erythric acid, 28.

Eiythritol, 28.

Eiythrose, 28.

Erythrocytes, 550.

Esox lucius, protamine in, 137.

Ether, cause of glucosuria, 81.

Ethereal sulphuric acids, 250, 251.

Ethyl alcohol. See alcohol.

Ethylamine carbonate, 234.

SUBJECT INDEX.

711

Ethylbenzene, 243.

Ethylene oxide, 113.

Ethylurea, 234.

Esters of monoamine acids, 172.

Evonymus, 56.

Exact science, relation to physiological
chemistry, 4.

Excitability, role of oxygen, 459.

Excrements, 534.

E.xpired air, 428.

Exudates, 578.

F.

Faeces, 534.

Fagine, 114.

Fasting values, 628.

Fat, as first assimilation product of
plants, 56; formation from sugar,
67; melting points, 103; assimila-
tion, 109.

Fats, 101-112; as source of energy, 338;
influence on protein requirement,
342 et seq.; on respiratory quotient,
346; on general metabolism, 347;
source of heat, 111; solvent of the
cells, 112; from carbohydrates, 303,
309; calorific value, 33.3; from pro-
tein, 323; influence on gastric secre-
tion, 500; on pancreatic secretion and
emptying of stomach, 527.

Fat, determination of, 325.

Fat, in secretions, 326; in faeces, 624; in
icterus, 519.

Fat metabolism, 623, 627, 631.

Fat migration, 327, 328.

Fattening, 345 et seq.

Fatty acids, from fat, 101; from lecithine,
112, 113; utilization in fat synthesis,
105; decomposition, 456.

Fatty degeneration, 327.

Fatty infiltration, 322, 327, 328.

Fatty tissue, 101, 110.

Fehling's test, 25.

Fellic acid, 516.

Fennentation, 27; reactions, 37, 38, 479.

Ferments, 73, 294, 295, 374,' 447, 461-
483. See Diastase, Lipase, Rennin,
Trypsin, Pepsin, Erepsin.

Fibrogenous substances, 537.

Fibrin, 133, 173, 535 et seq.

Fibrin ferment, 482, 537; zymogen of,
538.

Fibrin globulin, 542.

Fibrinogen, 133, 537.

Fibrinoplastic substance, 537.

Fibroin, 138.

Fictitious meal, 341, 498.

Ficus carica, 168.

Ficus macrocarpa, 168.

Figs, iron and lime content, 370, 380.

Fig-tree, proteolytic ferment from, 168.

Fish, protein from, 653.

Fish-scales, 1.38.

Flagellata, 52.

Flesh, composition of, 660. See Beef
and Meat.

Flies, larva;, 46; eggs, 326.

Floridese, 125.

Foetus, ash of, 369.

Foods, calorific value, 333; inorganic,
349-4.36; organic, 13; concept of,
353; requirement, 644-652.

Foodstuffs, consumption of, 647; replace-
ment of, 331-348; relative value of,
333, 334, 336.

Formaldehyde, 14, 56, 201, 446.

Formamide, 201.

Formic acid, 63, 74.

Frogs, extirpation of pancreas, 83 ; mucin
in the spawn, 141 ; function of
kidneys, 582. See also Mud-frog,
Nurse-frog, River-frog.

Fructose, 26, 27, 29, 38, 42, 66, 95, 190,
323, 471.

Fruit-sugar. See Fructose.

Fucose, 19, 24.

Functional nervous diseases, 615.

Fundulus heteroclitus, 359.

Fundus glands, 495.

Fungi, glycogen in, 47.

Furfurol, 21.

Fusel oil, 470.

G.

Galactanes, 31.

Galactitol, 44.

Galactonic acid, 28.

Galaetosaraine, 20, 35.

Galactose, 16, 20, 27, 28, 31, 38, 39, 40,
44, 148, 613; fermentation of, 471.

Gall-bladder, 516, 517.

Gas-absorption, law of, 413.

Gas-exchange, in formation of fats from
carbohydrates, 310-311; in the lungs,
428, 434 ; in the tissues, 435-138 ; in
work of kidneys, 584; under various
conditions, 663-670.

Gases in alveolar air, etc., 428-431.

Gas-secretion, in the lungs, 431 ; in
swimming-bladder of the fish, 433.

Gastric contents. See Chyme.

Gastric fistula, 164.

Gastric juice, 496-507.

Gastric lipase, 103, 497.

Gastropods, glycogen in, 46.

Gelatin, 137, 1 76 ; food value of, 215; blood
coagulation, 547.

Gelatin-sugar. See Galactose.

General metabolism, 620-662.

Generation, organs of, glycogen m, 47;
relation to other organs, 597-600.

Gentiobiose, 37.

Glands, activity seen under the micro-
scope, 465.

Glandular work, source of, 341.

Gliadin, 1.33, 173, 653.

Globin, 124, 141, 174, 397, 424, 556.

Globulins, 127, 132, 172.

Globuloses, 165.

Glomerulus Malpighi, 581.

Glucase, 60, 61.

Glucohemia, 77, 78, 85, 92.

712

SUBJECT INDEX.

Glucolytic ferment, 73, 447.

Gluconic acid, 57, 94.

Glucoproteids, 20, 141.

Glucosamine, 20, 31, 35, 94, 141, 159, 488.

Glucosazon, 26.

Glucose, d-, 16, 38, 39, 40, 66, 67, 190, 305,
323, 333, 336, 445, 471 , 480, 481 . See
also Grape-sugar and Dextro.se.

Glucose-ct-glucoside (Maltose), 481.

Glucose-/9-glucoside (Isomaltose), 481.

Glucosides, 19, 472.

Glucosuria, alimentary, 30, 76; starvation,
30; phloridzin, 30, 80; strychnine,
etc., 30, 80; after diabetic puncture,
77; injection of salt, 80, 359; extir-
pation of pancreas, 82.

Glucothionic acid, 49.

Glucurone, 32.

Glucuronic acid, 27, 31-34; oxidation in
diabetes, 94.

Glucuronic acid conjugates, 32-34.

Glutamic acid, 149, 304, 470, 653.

Glutamine, 151, 202.

Glutenin, 653.

Gluten casein, 132.

Glutin, 137.

Glyceric aldehyde, 28.

Glyceric acid, 28, 303, 470.

Glycerol, 14, 22, 28, 101, 112, .303, 314,
483.

Glycero-phosphoric acid, 113, 115.

Glycerose, 14, 28, 58, 302, 303, .304.

Glycine. See GlycocoU.

Glycine anhydride, 179, 186, 228.

Glycine-ethyl-ester, 181.

Glyco-apiose, 37.

Glycocholeic acid, 248, 514.

Glycocholic acid, 248. '

GlycocoU, 5, 147, 148, 229, 242-248, 278,
294, 458, 494, 514.

GlycocoU carbonate of calcium, 233.

Glycogen, .36, 44^9, 66-75, 315.

Glycol, 28, 113, 114.

Glycol aldehyde, 19.

Glycolose, 19, 28.

Glycolic acid, 28.

Glycosuria. See Glucosuria.

Glycyl-alanine, 474.

Glycyl-d-alanine, 187.

Glycyl-alanine anhydride, 186.

Glycyl-asparagine, 183.

Glvcyl-aspartic acid, 182.

Glycyl-glycine, 179, 180, 185, 217, 228,
474.

Glycyl-leucyl-alanine, 474.

Glycyl-phenyl-alanine, 179, 474.

Glycyl-i-tyrosine, 179, 184, 185, 187, 474.

Glyoxylic acid, 152, 162, 295.

Glyoxydiuride. See Allantoine.

Gmehn's test for bile pigments, 569.

Goats, rate of development, 371, 404.

Goat's blood, 554, 555.

Goat's milk, 366, 404, 405.

Goblet cells, 142.

Goitre region, 604.

Gold chloride solution, 357.

Gonionemus, 358.

Goose-blood, 559.

Goose-fat, absorption of, 117.

Gorgonia Carolini, 138.

Gossypose, 40.

Gout, 298.

Graham bread, iron and lime in, 370, 377.

Grains, protein in, 132, 133.

Grape, iron and lime content, 370, 380.

Grape-sugar, 13, 336, 358, 440. See also

d-glucose.
Grave-wax, 326.
Green-gage (or French) plums, iron and

lime content, 370, 380.
Grubs of insects, glycogen in, 46.
Guaiacum test, 446.
Guanidine, 156, 235.
Guanine, 22, 280, 283, 294, 297.
Guanylic acid, 22, 28.3.
Guinea pigs, experiments with, 64, 124,

368, 371, 393, 559.
Gulose, 17.
Gums, 42, 48.

H.
Haptophor groups, 685.
Hazel nuts, iron in, 381.
Heat, coagulation of proteins by, 121 ;

equivalent of work, 336, 424, 625;

measurement of, 335, 625; sources of,

75, 111, 342 ; regulation, 595, 643; rigor,

617.
Helianthus, inulin of, 29; fat cleavage,

103.
Helix, glycogen of, 46.
Helix pomatia, glucoproteid of, 144.
Helleborin, 21.
Hemal-lymph-glands, 573.
Hematin, 124, 141, 386, 392, 416, 558-

565.
Hematinic acids, 564.
Hematogen, 381, 387.
Hematoidin, 569.
Hematopoietic organs, 398.
Hematoporphyrin, 387, 396, 564, 573.
Hemicelluloses, 42.
Hemin, 396, 563, 565.
Hemochromogen, 416, 558.
Hemocyanin, 402.
Hemoglobin, 381; in new-born, 382-385;

crystals of, 125; behavior in high

altitudes, 436; spectroscopic behavior,

660; combination with oxygen, 415;

chemi-stry of , 55 1 -557 ; analysis of , 559 .
Hemoglobin formation, 387-392.
Hemoglobin-iron of new-born, 384, 385.
Hemolysis, 115, 551, 688-690.
Hemolysins, 689.
Hemophilia, 543.
Hemopyrrole, 564.

Hemp-seeds,albumincrystalsfrom,122,123.
Hen, hemoglobin of, 559.
Hen's egg, ash of the white, 369, 370, 380,

659; ash of the yolk, 369, 370, 659.

SUBJECT INDEX.

713

Henle's loop, 581.
Heptoses, 19.
Heredity, 670-676.
Herring, clupein in, 136.
Heterocyclic amino acids, 151.
Heteroxanthine, 297.
Hexahydrobenzene (inositol), 306.
Ilexobioses, 37.

High altitudes, effect of, 436.

Hippomelanin, 145.

Hippuric acid synthesis, 5, 50, 229,242,479.

Hirudin, 547.

Histidine, 154, 288.

Histidyl-histidine, 179.

Histones, 135, 174.

Hoffmann's tyrosine test, 151.

Holothuria, glycogen of, 46.

Homogentisic acid, 257, 271.

Horaothermous animals, 643.

Honey, ash of, 369, 370, 380.

Hordein, 653.

Horn, 138.

Horse, rate of growth, 371, 404; hemo-
globin, 559.

Horse-blood, 125, 418, 421, 554, 555.

Horse-milk, 366, 404.

Horse-tallow, 103.

Horny layer, 138.

Human beings, ash of infante, 368, 369;
rate of growth, 371, 404; iron con-
tent, 393, 394.

Human milk, ash of, 366, 369, 370, 377,
380, 404.

Humin substances, 146.

Himior, vitreous, 143.

Hunger. See Starv-ation.

Hydra, CO,, assimilation of, 52.

Hydrazones, 25.

Hydrobilirubin, 572.

Hydrochinon. See Quinol.

Hydrochloric acid, 203, 204, 219, 220,
490, 492, 496, 523.

Hydrocyanic acid, 18, 220, 238.

Hydrogen, 200, 438.

Hydrogen peroxide, 313, 449.

Hydrolysis of proteins, 146, partial, 186.

Hydro- p-cumaric acid, 254.

Hydrosol, 120.

Hydro.xyiamine, 201.

Hydroxymandelic acid, p-, 170, 254.

Hydroxynaphtylamine, 446.

Hydroxyphenyl-a-aminopropionic acid,
151.

Hydroxyphenyl-ethylamine, 259.

Hydroxyphenylacetic acid, 170, 254, 255,
273.

Hydroxyphenyl-propionic acid, p-, 170,
254, 255, 259, 273.

Hydroxyprolin, 151.

Hydroxypyrrolidin-carboxylic acid, 151.

Hydroxy- ^-quinolin-carboxylic acid, p-,
153.

Hydroxy-benzoic acids, 252.

Hydroxybutyric acid, ,5-, 97.

Hydroxy-fatty acids in fats, 102.
Hydroxy-quinolin-sulphate, 252.
Hydroxy-/)-cumaric acid, 254, 255.
Hyoglycocholic acid, 515.
HyperglucEemia. See Glucohemia.
Hyperisotonic solution, 550.
Hypersecretion of gastric juice, 508.
Hypisotonic solution, 550.
Hypophysis, 609.
Hyposulphurous acid, 249.
Hypoxanthine, 280, 292, 297.

Ichthulin, 144.

Ichthylepidin, 138.

Idose, 17.

Icterus, 518, 570.

Ileum, absorption of iron in, 389.

Illuminating gas, 81.

Imadazole, 284.

Immunity, 509; of mucous membrane,

494; acquired, 680.
Inactivated serum, 688.
Indican, 259.
Indigo, 201.
Indigo-blue, 258.
Indigo-red, 259.
Indigotate, sodium sulph-, 582.
Indirect conclusions, 4, 307, 313, 314.
Indirubin, 259.
Individual variations, 8.
Indole, 153, 170, 251, 258, 457.
Indophenol, 446.
Indoxyl, 201, 252, 257, 457.
Indoxyl-gl neuronic acid, .32.
Indoxyl-sulphuric acid, 252.
Indoxyl-sulphate of potassium, 252.
Infants, ash of, 368, 369; rate of growth,

371, 404.
Inorganic foodstuffs, 349 et seq.
Insects, glycogen in the grubs of, 46.
Inspired air, volume of, 429.
Internal secretion, 90.
Innervation, of the liver, 77; of the lungs,

434 ; of the kidneys, 585.
Inositol, 93, 306.
Inorganic foods, 349-407.
Insect blood, 125, 447.
Inspired air, composition of, 428.
Intestinal digestion of carbohydrate, 60;

of fat, 104 ; Qf protein, 208.
Intestinal flora, 62, 64, 218.
Intestinal gases, 438.
Intestinal juice, 512 et seq.
Intestinal putrefaction, 218.
Intestinal respiration, 438.
Intestine, absorption of iron in small,

388 et seq. ; digestion and absorption,

51 1 et seq. ; elimination of iron in the

large, 388 et seq.
Inulase, 482.
Inulin, 29, 41, 44, 95.
Inversion, 38.

714

SUBJECT INDEX.

Invertase, 467, 482, 512.

Invertin, 61, 461.

Invert-sugar, 29, 38.

Iodine, 41, 406, 607.

lodothyrin, 607, 608.

Ions, 358, 359, 592.

Iridete, 44.

Iron, 380-402.

Iron, bacteria, '194.

Iron content of foodstuffs, 380, 381 ; of

rabbits, 681 ; guinea pigs, 682.
Isoamylalkohol, 149, 455, 470.
Isobilianic acid, 515.
Isobutyric acid, 455.
Isobutyl-acetic acid, 455.
Isocholesterol, 117.
Isodynamics, law of, 331, 336.
Isolactose, 38, 480, 481.
Isoleucine, 148.
Isomaltose, 37, 479-181.
Isomers, behavior in hydrolysis, 471-476.
Isotonic solutions, 550.
Isovaleric aldehyde, 149-455.
Isovalerianic acid, 455.
Isovalero-nitrile, 149.
Ixodis-ricinus, 547.

Jalap, rhodeose in, 19.

Jecorin, 49.

Jejunum, iron absorption in, 389 et seq.

Jequirity seeds, 682.

Johannisbrot tree, cane sugar in, 38.

Kephir lactase, .38, 480.

Keratins, 138, 176.

Ketoses, 19.

Kidneys, glycogen in, 47; glucothionic

acid, 49; elimination of sugar, 82;

hippuric acid, 246, 247; elimination

of iron, 388; of lime, 392, 397;

function of, 579-594.
Kynurenic acid, 153, 260.
Kyrins, 177.

Laborer, diet of, 645, 646.

Laccase, 447.

Lactarius volemus, 29.

Lactase, 323, 482, 513.

Lactates, oxidation of, in diabetes, 93.

Lactation, 386.

Lactic acid, 238, 303, 304; from carbo-
hydrates in the intestine, 63; as
intermediate product in alcoholic
fermentation, 483.

Lactic-acid-ferment, 482.

Lactic-acid fermentation, 27, 469.

Lactobiose, 39.

Lacto-glucase, 38.

Lactose, 39.

Laevulic acid, 282, 30G, 313.

Lffivulic aldehyde, 306, 313.

Laked blood, 550.

Lampyris splendidula, 411.

Langerhans cells in the pancreas, 90,

93.
Lanolin, 105.
Large intestine, elimination of iron in,

389 et seq.
Latent period of the stomach, 498.
Lathra?a squamaria, 122.
Laurie acid, 102.
Lavosin, 44. .

Laws, of isodynamics, 331-348 ; of conser-
vation of energy, 334 et seq. ; of the
minimum, 356; of gas-absorption,
413; of specific sense energy, 494.
Lecithides, 115.

Lecithin, 112-116, 403; content in cer-
tain organs, 114; cleavage by lipase,
115; activatos, 115; in nervous
tissue, 613.
Legumin, 132.
Leguminoses, 196.
Legumes, legumin in, 132.
Lens, crystalline, 138.
Lentils, iron content, 381.
Leptothrix ochracea, 194.
Leucaemia, 290.

Leucine, 148, 239, 304, 305, 325; decom-
position of the d-l form in the organ-
ism, 453, 454; cleavage by fungi, 470;
cleavage by yea.sts, 470; taste, 494.
Leucine-ethyl-ester, 475.
Leucine carbonate of calcium, 233.
Leucinimide, 169.

Leucocytes, glycogen, 47; uric acid, 389;
in blood coagulation, 540; in general,
551 et seq.
Leucyl-alanine, 179, 474.
-alanyl-alanine, 179.
-asparagine, 183.
-aspartic acid, 182.
-glycine, 179, 474.
-glycyl-glycine, 179, 181, 474.
-isoserine, 474.
-leucine, 179, 186, 228, 474.
-proline, 179, 474.
-tetraglycine, 179.
-i-tyrosine, 181, 474.
Levulose, 27. See rf-Fructose.
Lichenin, 44.
Lichens, 53.

Lieberkiihn's glands, 512.
Licbermann's reaction for proteins, 162.
Life without bacteria, 64.
Ligamentum muchse, 138.
Lignification, 42.
LiliaceEe, 44.
Limax, 46.
Limonene, 118.
Lipoids, solubility of, 112.
Lipase, 103, 475, 482; of stomach, 429;

of intestinal juice, 512.
Lithium, 407.

SUBJECT INDEX.

715

Liver, glycogen, 47; glucothionic acid
49; jecorin, 49; glycogen storehouse
66; carbohydrate metabolism, 77
behavior after pancreas extirpation
84, 636; in diabetes mellitus, 03
urea formation, 231; uric acid, 237
metabolism in diabetes, 353; iron
depot, 391; bile, 513-521; relation
to fibrinogen, 548; part played in
formation of bile-pigments, 569,
570.

Liver atrophy, 264.

Liver-bile, 516.

Liver-proteid, 22.

Loach, 438.

Localization of combustion, 409.

Luminescence, 53.

Lump-fish, protamine from, 137.

Lung arteries, 428, 429.

Lung catheter, 429.

Lungs, gas exchange in, 408-432;
metabolism in, 431, 441; as glands,
431; surface of, 428; reducing power,
431 ; glycogen in, 47.

Lung veins, 42S.

Lupines, 133, 150.

Luteins, 549.

Lymph, 573-578.

Lymphagogues, 576.

Lymph fistula, 108.

Lymph glands, function of, 577.

Lymph vessels, glycogen in, 47.

Lysine, 154.

Lysyl-lysine, 179, 259, 305.

M.

Mackerel, scombrin in, 136.

Madder, 21.

Maggots, sugar formation by, 319.

Magnesium, 402.

Magnesium salts, from albumin. 123.

Malt, .39.

Maltase, 479, 480; of intestintd juice,

512.
Maltobiose, 39.
Maltoglucase, 37.
Maltose, 37, 39, 46, 59, 96, 480.
Malt-sugar, 39.

Mammary glands, 49, 595, 596-599.
Man. See Human beings.
Mandelic acid, 470.
Mandelo-nitriie-glucoside, 20, 480.
Manganese, 402.
Manganous oxide, 450, 451.
Manganese peroxide, 450, 468.
IMannan^ 29.

Manna tetrasaccharide, 40.
Mannitol, 27, 93.
Manuonic acid, 28.
Mannonic-acid-lactose, 470.
Manno-rhamnases, 36, 190.
Mannose, 16, 26, 28, 29: transformation

into glucose and fructose, 67, 190;

fermentation of, 471.

Mannose hydrozone, 26.

Manno-saccharic acid, 28.

Mariotte's law. See Boyle's law.

Marrow of bones, 612.

Meal worm, 123.

Meat, ash of, 366, 370, 377, 381; influ-
ence on gastric secretion, 502; in
pancreatic secretion, 531 ; composi-
tion of, 660.

Meat broth, influence on gastric secretion,
500.

Meat, consumption of, by different races
of people, 659.

Meat juice, influence on gastric secretion,
500.

Meconium, 218.

Medulla oblongata, 77.

Melanins, 144.

Melanose, 447.

Melanotic sarcoma, 144.

Melibiase, 482.

Melibiose, 37.

Melitriose, 37, 40.

Melons, 168.

Membranse propria?, 138.

Menstruation, 398.

Mercaptan, 494.

Mercapturic acid, 159-160.

Mercury salts, 357.

Mesitylene, 458.

Mesitylenic acid, 458.

Mesoporjjhrine, 564, 567.

Meso-tartaric acid, 452.

Mesoxvl-urea, 279.

Metabolism, 620-662; of cells. 49; with-
out salts, 354, 356; influence of work
upon, 626; effect of bodv surface,
628-6.30; influence of age, 630; in
starvation, 6.30; during pregnancy,
642; external conditions, 643; in
osteomalacia, 377.

Metal-albuminates, 128.

Methemoglobin, 125, 425, 562.

Methane, 63, 438.

Methods, value of, 11.

Methylamine, 602.

Methylene blue, 411.

Methyl-glucosides, 472-473.

Methyl-2-6-dihydroxy-pvrimidine, 281.

Methylfuran, 306.

MethylglycocoU, 235.

Methylguanine, 7- (epiguanine), 298.

Methylquanidine-acetic acid, 235.

Methyl imidazole. 286.

Methyl indole, 257, 602.

Methyl pentosans, 23-65.

Methyl pentoses, 19.

Methyl propyl-pyrrole, 564.

Methyl quinol, 252.

Methyl quinolin, 457.

Methyl uracil, 281.

Methyl xanthine, 297.

Methyl xyloside, 473.

Mice. See Mouse.

Micro-chemical tests, 105, 390.

716

SUBJECT INDEX.

Migration of material, in salmon, 351, 352;
in starvation, 352.

Milk, milk-sugar in, 39; ash of, 366; lime
in, 370; influence on gastric secretion,
500, 502, 503 ; influence on pancreatic
secretion, 531 ; composition of, 654;
relation of composition to rate of
development of suckling, 367, 370,
371, 404, 655.

Milk-albumin, 132.

Milk-globulin, 132.

Milk-sugar, 13, 31, 37, 39, 323; digestion
of, 62; calorific value, 333; action on
muscle, 358.

Millon's reagent, 151.

Mimicry, 677.

Molecules, size of, starch, 41 : proteins, 128.

Molisch's sugar test, 162.

Mollusks, 46, 402.

Mono-amino-di-carbo-oxylic acid, 150.

Mono-amino-mono-carbo-oxylic acids, 148,
151.

Mono-amino-hydroxy-carbo-oxylic acids,
149, 151.

Mono-amino-acids, 129.

Mono-methyl-xanthine, 297.

Mono-sodium urate, 299, 593.

Monosaccharides, 19-21.

Moore's sugar test, 25.

Morbus Addisonii, 603.

Morbus Basedowii, 609.

Morphine, 80, 677.

Moss, Iceland, 44.

Mouse, iron content of a, 393.

Mouth, 485.

Mountain sickness, 437.

Mucins, 141, 142.

Mucin-like substances in urine, 268.

Mucic acids, 28, 39, 94.

Mucoids, 142.

Mucous glands, 485.

Mud-frog, sterile larvje, 64.

Muroena, 546.

Musca lucilia, 478.

Muscarine, 114.

Musca vomitoria, 319.

Muscle-albumin, calorific value, 3.33,
3.36.

Muscles, action of certain salts upon,
358; creatine in, 235; function of,
611.

Muscles, glycogen, 46; jecorin, 50; glyco-
gen stores, 67; behavior after extir-
pation of pancreas, 85; consumption
of carbohydrates, 87; purine bases,
292.

Muscular force, source of. 337.

Mussels, conchiolin of, 138.

Muscular work, 67; influence on albumin-
fattening, 618.

Muscular stomach of birds, 138, 507.

Mutual relations of fat, carbohydrate and
protein, 301-348, 3.58 et seq.

Mutton-tallow, melting-point, 103; absorp-
tion of, 108; assimilation, 110.

Myogen, 133.

Myosin, 133.

Myrostic acid, 102.

Myrosin, 20, 461.

Myxcedema, 605.

My.xomycetes, oxygen requirement of, 459.

N.

Naphthol, a, 162, 446.
Naphthoic acid, 229, 243.
Naphthuric acid, 229, 243.
Navel. See Umbilical cord.
Naphtylamine, a, 446.
Nepente, 168.
Nephritis, 268.
Nerves, facial, 485.

glossopharyngeal, 485, 494.

lingual, 486.

splanchnic, 78.

sympathetic, 485, 507, 527, 603.

trigeminal, 495.

vagus, 78, 4.34, 498, 521.

degeneration of, 616.
Nervous diseases, 615, 617.
Nervous system, influence on liver,

77.
Nervous tissue, phosphorus content, 403;
function of, 611 to 614; affinity to
tetanus toxin, 686.
Neurine, 114.
Neurokeratin, 1.38, 613.
Neurosis, 459.
Neutral fats, 101.
Nitrates, 193.
Nitrification, 193.
Nitrites, 194.
Nitric acid, 193.
Nitric-oxide-hemoglobin, 561.
Nitro-benzaldehyde, 244, 457.
Nitro-benzene, 81.
Nitro-benzoic acid, 244.
Nitro-benzyl alcohol, 34, 458.
Nitro-hippuric acid, 243.
Nitro-phenol, 252.
Nitro-toluene, 34, 458.
Nitrogen, 119, 194-200, 4.38.
Nitrogen content of blood, 415; of fpeces,

624.
Nitrogen in soil, 196.
Nitrogenous equilibrium, 342, 637-640.
Nucleases, 288.
Nucleic acids, 144, 275, 276, 300; from

yeast, 21 ; digestion, 288.
Nuelein bases. See Purines.
Nucleic-acid-protamine, 140.
Nucleins, 141, 275, 387, 388, 403.
Nucleoalbumins, 133, 173, 397, 403.
Nucleohistone, 139.
Nucleoproteids, 140, 275-300.
Nurse-frog, 353.

Nutritional value of foods, 621-626.
Nutrition without salts, 354 et seq.
Nuts, conglutin, 133; fats from carbo-
hydrates, 308.

SUBJECT INDEX.

717

0.

Oats, gliadin in, 133.

Obesity, HI.

Ocean, denitrifying bacteria in, 199.

Octodecyl alcohol, 102, 595.

CEsophageal fistula, 341.

CEsophageal groove, 509.

Oil as first assimilation-product of plants,
56.

Oil-bag of birds, 102, 595.

Oil of Pennyroyal, English, 327.

Oil-plasma, 545.

Oils, ethereal, 303.

Oil-seeds, fats from carbohydrates, 308
et seq.

OleaciE, 56.

Oleic acid, 102, 113, 303.

Oleum Pulegii. See Oil of Pennyroyal,
English.

Olive oil, 108.

Olives, carbohydrate and fat in, 308.

Opalins, glycogen in, 46.

Opalisin, 654.

Optical activity, 15 et seq.

Oranges, iron and lime in, 370, 380.

Orcin, 252.

Organ pentoses, 22.

Organs, experiments in surviving, 9;
hematopoetie, 398.

Organs of generation, 597 et seq.

Organs of phosphorescence, 411.

Ornithine, 155, 169, 227, 228, 245.

Omithuric acid, 155, 245.

Osazones, 26.

Oscillaria sancta, 671.

Osmosis, 357.

Osseoalbumoid, 138.

Osteoidal tissue, 374.

Osteoclasts, 612.

Osteomalacia, 377.

Oteoplastic tissues, 374.

Osteoporosis, 375.

Oval, 433.

Ovalbumin. See Egg albumin.

Ovaries, 379, 599.

Ovimucoid, 143.

Ovokeratin, 138.

O.xalic acid, 28, 279, 295, 444, 457.

O.xalylurea, 279.

O.K-blood, 421.

Ox-muscle, 653.

Oxidases, 445, 482.

Oxidation, animal, 409, 439-460.

Oxidation ferments, 445 et seq.

Oxidation power, 93.

Oxidation processes, 74, 411, 452.

Oxidizable substances, 408.

Oxy-. See Hydroxy.

Oxydases, 445, 482.

Oxygen, 408—438; free in saliva, 411 ; actu-
ation, 442.
absorption by the blood, at different
temperatures, 417; at different pres-
sures, 418; influence of CO,, 427.
capacity of the blood, 427.

Oxygen, carriers of, 445.

consumption by animals of different

size, 629.
supply of foetus, 410, 411; of insects,

411, 412.
tension of lymph, 435.
tension curves, 418.
O.xygenases, 449.

O.xyhemoglobin, 126, 127, 141, 558-561.
Oxyneurine, 114.
Oxyproteic acid, 270.
0.xypurine, 6, 280.
Oxysantonine, 457.
Ozone, 312, 441.
Ozonide, 313.

P.
Palms, cellulose stores in, 44; cane-sugar

content, 38;
Palmitic acid, 303 ; from lecithin, 113 ; from

fat, 102.
Palmitic-acid-myricyl ester, 102.
Pancreas, nucleic acid in, 285; carbohy-
drate metabolism, 82, 83, 87; part

played in digestion, 513 et seq. ;

glycogen, 47; glucothionic acid, 49.
Pancreas, extirpation of, 82.
Pancreas-nucleoproteid, 21.
Pancreatic fistula, 164.
Pancreatic juice, action on nucleic acids,

288; ab.sorption of fat, 107; general

function, 521 et seq.
Papayotin, 168.
Papillionacse, 197.
Parabanic acid, 279.
Paracasein, 207.
Parachymosin, 207.
Paraffin plasma, 545.
Paralyzers, 468.
Paramucin, 142.
Paranuclein, 1.34.
Para nuts, 123.

Parathyroid glands, 604, 605, 606.
Paraxanthine, 297.
Parotid gland, 484 et seq.
Parthogenesis, 360.
Partial pressure, 374.
Partial hydrolysis of proteins, 186.
Patella, 402.
Pathology, relation to physiological

chemistry, 3.
Paunch, 509.

Pears, iron and lime content, 370, 380.
Peas, ash of, 369, 377, 388.
Pectases, 482.
Pecten irradians, 148.
Peetinase, 482.

Penicillium glaucum, 470, 472.
Pentaglycine, 179.
Pentamethyldiamine, 154, 170, 259.
Pentosans, 23, 42, 65.
Pentoses, 19, 21, 323.
Pentoses in organs, 22; in food, 24.
Pentosuria, 23, 96.
Pepsin, 203, 204, 462, 482, 499.
Pepsin digestion, 163.

718

SUBJECT INDEX.

Pepsinogen, 497.

Peptides. See Polypeptides.

Peptone blood, 546.

Peptones, 165, 178, 188, 204, 208, 546.

Perearbonic acid, 57.

Perforating fibers, 612.

Periosteum, 612.

Peroxide formation, 449, 4.50.

Peroxydases, 449.

Persea-gratissima, 29.

Perseitol, 29.

Petroselinum apiin, 36.

Phenaceturic acid, 243, 256.

Pharmacology, relation to physiological
chemistry, 677.

Phenol, 170, 229, 252, 457, 458.

Phenylacetic acid, 170, 244.

Phenol-glucuronic acid, 32.

Phenolphtalein, 446.

Phenolphtalin, 446.

Phenol-sulphate of potassiimi, 229, 252,
457.

Phenol sulphuric acid, 252, 254.

Phenylalanine, 151, 251, 256, 259, 272, ,304.

Phenylaminopropionic acid, 151.

Phenyldiaminine, 446.

Phenylethylaraine, 259.

Phenylhydrazine, 25.

Phenylpropionic acid, 170.

Phloretin, ,30, 82.

Phloretic acid, 30.

Phloridzin, 21, .30, 81.

Phloridzin poisoning, 31, 327, 587.

Phloroglucinol, 30.

Phospho-globulin, 137.

Phosphoglucoproteid, 44.

Phosphorescent organs, 411.

Phosphoric acid, 22, 112, 277.

Phosphoric acid amide, 284.

Phosphorus, 403-406; behavior in organ-
ism, 403, 444; content of ner\'ous
tisisue, 613.

Phosphorus poisoning, 30, 81, 265, 327,
328, 548.

Phrynolysin, 690.

Phycocyan, 125.

Phyllopoqihyrin, 568.

Physcia parietina, 217.

Physiology, relation to physiological
chemistry, 3, 484, 485.

Physiological salt solution, 550.

Phytocholesterol, 116.

Phytovitellin, 132, 173.

Pigment formation in Morbus Addisonii,
603.

Pigments, 569.

Pigs, development of, 370, 404.

Pig's blood, iron and lime content, 370,
381; analysis of, 554, 555; hemo-
globin from, 559.

Pig's fat, absorption of, 108.

Pig's milk, 366, 404, 405.

Pike, protamine from, 137.

Pinene, 188.

Pine-seeds, albumin from, 173.

Pinguicula, 168.

Pinna squamosa, 402.

Planarians, 52.

Plant casein, 173.

Plant crytalloids, 122.

Plasma, 415, 421, 422, 423, 535.

Plastein, 208.

Platinum catalysis, 468.

Pleurobranchia Meckelii, 490.

Plums, iron and lime content of, 370, 380.

Pneumonia, 350.

Poikilothermous animals, 643.

Polydypsia, 83.

Polypeptides, 17S, 179, 183-186, 192, 474,
475.

Polyphagia, 83.

Polyps, 46.

Polysaccharides, 19, 36-49.

Polyuria, S3.

Potash, content of certain foodstuffs, 366.

Potatoes, ash of, 369, 370, 380.

Precipitin formation, 668.

Preserved yeasts, 464.

Primula, 29.

Principal cells, 495.

Problems of physiological chemistry, 1-12.

Proferments, 465.

Proline, 151, 152.

Prolyl-leucine, 179.

Propionic acid from alanine, 170.

Propylbenzene, 243.

Prosecretin, 528.

Prostate gland, 599.

Protagon, 613.

Protamines, 135-137.

Protective substances, 23.

Proteid of the liver, 22.

Proteids, 131, 139, 275. See also Proteins.

Proteins, 119-275.

Protocatechuic acid, 252, 254; from
adrenalin, 602.

Protococcus vulgaris, 28.

Proton, 226.

Protoplasm, 456, 672.

Protozoa, 46.

Psalterium, 509.

Pseudomucin, 142.

Pseudonucleins, 134.

Ptyalin, 59.

Ptyalose, 39.

Pumpkin seeds, 122.

Purine bases, 277, 278, 289-294; decom-
position of, 292-297. ^

Purine ring, 279.

Purine value, exogenous and endogenous,
291.

Pus, 578.

Pus cells, 140.

Putrefaction, in the intestine, 253; of
protein, 218.

Putrefactive products of proteins, 169,
170..

Putrescine, 155, 170, 260, 267.

Pylorus, function of, 507.

Pj'ridine, from adrenalin, 602.

SUBJECT INDEX.

719

Pyrimidine bases, 276.

Pyrimidine ring, 281.

Pyrocatechol-sulphuric acid, 251.

Pyrogallol, 252, 440.

Pyrotartaric acid, 169.

Pyrrole, from adrenalin, 602; in Morbus

Addisonii, 603.
Pyrrolidine carboxylic acid, 151.
Pyrrole reaction, 152.

Q.
Quantity, concept of, 11.
Quercitrin, 24.
Quinol, 253, 4.58.
Quinoyl sulphuric acid, 254.
Quotient, respi ratory, 346, 627 ; D -- N, 321 ,
322.

R.

Rabbits, ash of young, 368; rate of

development, 370. 404; blood of,
554, 555; composition of milk, 366,

404; hemoglobin content, 383; iron

content, 39.3.
Racemic acid, 453.
Racemic bodies, amino acid in, 181.
Rachitis, 371 et seq.
Raffinose, 37, 40.
Rape-seed oil, 1, 10.
Raspberries, iron and lime content, 370,

380.
Rats, hemoglobin of, 383.
Reaction, biological, 668 et seq.
Reactions, of the hexoses, 25-27.
Reabsorption, in urinary tubes, 584 et

seq.
Receptors, 683.

Reduction-power of the lungs, 431.
Reduction processes, 411, 442.
Reflex action, 490.
Regeneration, 674.
Regulation, mechanism of, 436.
Regurgitation, 509.
Reindeer, milk of, 654.
Relation of foodstuffs, 2, 5 et seq, 301

et seq.
Relation of milk compositions to rate of

development, 366.
Relative value of foodstuffs, 333, 334,

336.
Renal calculi, 297.
Rennet bag, 509.
Rennin, 205-207, 482, 495, 497.
Reproductive glands, glycogen in, 47.
Reproductive organs, 597 et seq.
Reserve air, 429; carbohydrate, 42;

cellulose, 44.
Residual air, 429.
Resistance capacity of cell-substance

towards ferments, 191.
Respiration, internal and external, 412;

anaerobic, 412; cutaneous, 432-437.
Respiration calorimeter, 625.
Respiratory quotient, 346.
Retention cysts, 487.
Reticulin, 138.

Reticulum, 509.

Reversible fermentation reactions, 37, 479-

483.
Rhamninose, 40, 190.
Rhamnose, 19, 24, 29, 40, 190.
Rhamnus infectoria, 40, 190.
Rhinantaca?, 197.
Rhizapoda, 46, 459. »

Rhodeose, 19.
Rhodophyceoe, 125.
Ribose, 29.

Rice, iron and lime content, 370, 380.
Ricin, 681.

Ricinus communis, 681.
Rigor of death, 133, 617, 618; of heat,

617.
River-crabs, tyrosinase of, 447.
Roman-snail, 144.
Root-nodules or tubercles, 196.
Ruberj-thric acid, 21.
Rubia tinctorum, 21.
Rumen, 509.
Ruminants, 509.
Rumination, 509.
Rye, 133, 369, 370, 380.

Saccharin, 318.

Saccharic acid, 27; d-, oxidation in dia-
betes, 94.

Saccharobiose, 38.

Saccharo-coUoids, 41.

Saccharomyces apiculatus, 471.

Saccharomyces intermedians, 481.

Saccharomyces productivus, 471.

Saccharose, 38.

Salicin, 21.

Salicylic acid, 243, 440, 446.

Salicylic aldehyde, 94, 440, 446.

Salicyhc amide, 252.

Saliva, 203, 485-494; action on carbo-
hydrates, 59 ; oxygen content of, 410.

Salivary glands, function of, 485-494 ;
microscopic appearance, 487, 488;
mucin in, 142.

Salmine, 1.36, 175.

Salmo fario, protamine in, 137.

Salmonucleic acid, 285.

Salmon, migration of matter in, 1.30, 287,
351.

Salmon testes, histon, 135; salmine, 136.

Salmon spawn, 140.

Salsola, or Salt-wort, 364.

Salt, common. See Sodium chloride.

Salt, preparation of substitute for, 363,
364.

Saltpeter, formation in soil, 193.

Salts, 349 et seq. ; storage of, by foetus,
375.

Salts, action of, upon muscle, 357 et seq.

Salts, requirement of, by adults, 366; by
growing individuals, 366; by suck-
lings, 376.

Santonine, 457.

Saponification, 103.

720

SUBJECT INDEX.

Saponin, 21, 116.

Sapotoxin, 31.

Sarcolemma, 138.

Sarcoma, 675.

Sarcosine, 235, 660.

Schweitzer's reagent, 41.

Sciences, e.xact, relation to physiological
chemistry, 4.

Scombrine, l56, 174.

Scybala, 534. .

Scyllium catulus and canicula, S3.

Scymnol, 515.

Scymnol-sulphuric acid, 515.

Scymnus borealis, 513.

Sea-moss, 19, 24.

Sea-urchin, 140.

Sebaceous glands, 595.

Secalin, 44.

Secretin, 527 et sen., 596 et seq.

Secretion, internal, 596 et seq.

Sedimentum lateritium, 573, 592.

Selachia, 225.

Seminase, 482.

Seralbumin and Serglobulin. See Serum
albumin and Serum globulin.

Serine, 149, 239, 304.

Serous membranes, 577.

Serum, 536.

Serum albumin, 131, 132, 172, 548;
crystals, 124, 127; glutamic acid con-
tent, 653.

Serum globulin, 131, 132, 172, 548; glu-
tamic acid content, 653.

Seryl-serine, 179.

Sexual character, secondary, 599.

Sexual organs. See Regenerative organs.

Schweigger-Seidel's urinary tubule, 581.

Sheath-fish, protamine in, 137.

Sheep, rate of development, 370, 404;
blood, 554, 555; milk of, 360, 404,
405.

Side-chains, 683.

Side-chain theory, 682-691.

Silicate of soda, 120.

Silicic acid, 120.

Silk, 139; -fibroin, 176; -gelatine, 176.

Silver, absorption by the cells, 357.

Silurus glanus, 137.

Sinapis alba, 57.

Size, influence upon metabolism, 628

Skatole, 153, 170, 201, 251, 257, 458.

Skatole-acetic acid, 153, 170, 259.

Skatole-amino-acetic acid, 153, 258.

Skatole-carboxylic acid, 153, 170, 259.

Skatoxyl, 257, 458.

Skatoxyl-glucuronic acid, 32.

Skatoxyl-sulphuric acid, 251.

Skin, 432-437.

Small intestine, 389-392, 511 et seq.

Smell, 489-495.

Snail, mucin from, 142.

Snail, Roman, 144.

Snails, acid, 490.

Snake venom, 690.

Soaps, 103 ; influence on pancreas, 526.

Soda, content of foods, 366.

Sodium bicarbonate, 423.

Sodium carbonate, 423.

Sodium chloride, 361-365; glucosuria
produced by, 79, 80; cycle of, 525.

Sodium phosphate, 423, 593.
silicate, 120.
sulphindigotate, 582.
urate, 299, 593.

Soil, nitrogen in, 196.

Soja hispida, 197.

Sol, 120.

Solanin, 116.

Soldiers, diet of, 647.

Sorbite, 27, 448.

Sorbose, 448.

Species, conception of, 663-678.

Specific composition of body-cells, 329,
330.

Specific nature, of oxydases, 446; of
ferments, 467.

Spectroscopic behavior, of oxyhemoglobin,
560; hemoglobin, 560; carbonic-oxide-
hemoglobin, 561 ; nitric oxide hemo-
globin, 561 ; sulph-hemoglobin, 562.

Spermaceti, 102, 105, 108.

Spermatozoa, 135, 140, 285.

Sperm-whale, 102.

Sphferochinus granulans, 123.

Sphingosin, 20, 31, 613.

Spider poison, 648.

Spinach, iron in, 381, 388.

Spirogyra, 357.

Spleen, glucothionic acid, 49; jecorin, 49;
iron depot, 390; influence on pan-
creas, 531; on blood-formation, 573;
function of, 610, 611.

Splitting-processes. See Cleavage and
Hydrolysis.

Sponge, 46, 138.

Spongin, 1.38.

Squirrels, hemoglobin, 125, 559.

Stacchyose, 40.

Standard calorific values of foodstuffs, 337.

Staphylosin, 690.

Starch, 13, 36, 42; soluble, 43; calorific
value of, 333, 336; paste, 43.

Starvation, glucosuria, 30; effect on
diabetic puncture, 77; migration of
matter during, 352; disappearance of
glycogen, 68; metabolism, 631 ; rela-
tion of separate organs in, 635.

Steapsin. See Lipase.

Stearic acid, from fats, 102; from lecithin,
113; formation of, 303.

Stereochemistry, of sugars, 16-18; of poly-
peptides, 180, 181, 184, 185.

Stereoisomers, decomposition of, fin the
organism, 452; relation to cleavage,
470-476.

Stereobilin, 534.

Stomach, function of, 495-510; small,
496; fistula, 496; extirpation of, 509;
emptying of, 507.

SUBJECT INDEX.

721

Strophantin, 37.

Strophantobiose, 29.

Strychnine, glucosuria, 30, 81 ; influence

upon the nervous system, 459.
Strawberries, 370, 377, 381.
Strawberry extract, 577.
Sturgeon, sperma of, 137; sturin in, 136.
Sturin, 136, 175.
Sublimate, corrosive, SI.
Substance, fibrogenous and fibrinoplastic,

537.
Sublingual and submaxillary glands, 485-

489.
Succinic acid, 170, 483; oxidation of,

during diabetes, 94.
Sucklings, ash of, 368.
Sugar, 370, 380.
Sugar acids, 27, 28.
Sugar-beet, 38.
Sugar center, 77.
Sugar-cane, 38.

Sugar-content of the blood, 29.
Sugar-maple, 38.
Sugar-millet, 38.

Sugar puncture. See Diabetic puncture.
Sugars, simple, 19 et seq. ; compovmd, 36

et seq.; formation from fat, 310-322;

formation from protein, 316-321.
Sulphanilcarbamic acid, 234.
Sulphanilic acid, 234.
Sulph-hemoglobin, 561.
Sulphocyauic acid, 250.
Sulphur, 119, 194, 249, 406.
Sulphur bacteria, 194.
Sulphureted hydrogen, 170, 194, 401, 438.
Sulphuric acid, 249, 457, 490.
Sulphurous amino acids, 156.
Sunflower-seeds, 123.
Suprarenal gland, 600-003.
Surface-tension, 532.
Sur\nving organs, experiments with, 9.
Sweat-glands, 595.
Swimming-bladder of fishes, 433.
Symbiosis, 53.
Synovial fluid, 578.
Synthesis, of fat in intestinal wall, 105;

of polypeptides, 179; by ferments, 37,

38, 479-483.
Syntonin, 336.

T.
Takadiastase, 38.
Talose, 18, 472.
Tape-worm, glycogen, 46.
Tannin, 252, 305.
Tartaric acids, 28, 94, 453, 470.
Tartronic acid, 28, 239.
Taste, 489-195.
Taste-buds, 494.
Taurine, 158, 234, 248, 515.
Taurochenocholic acid, 515.
Taurocholic acid, 158, 248, 515.
Teeth, 493.
Temperature, during w-ork of salivary

glands, 486 ; of kidneys, 584.
Tendon mucoid, 143.

Tension, of CO,, 421; of oxygen, 418.

Tenebrio molitor, 123.

Terpenes, 117, 307.

Testudo graeca, 433.

Testes, 123, 600; of a bull, 285.

Tetanolysin, 690.

Tetanus toxin, 116, 687-690. ■

Tetraglycine, 179.

Tetraglycyl-glycine, 474.

Tetraniethylenediamine, 155, 170, 259.

Tetrasaccharides, 40. ,

Tetroses, 19, 37.

Theobromine, 200, 280.

Theophylline, 2S0.

Thiolactic acid, a, 169.

Thiophenaldehyde, 244.

Thiophenuric acid, 244.

Thiosulphuric acid, 249.

Thread bacteria, 194.

Thread fungi, 198.

Thujone, 34.

Thujone hydrate, 34.

Thymine, 281, 298.

Thymol, 162, 252.

Thymus glands, histon, 135, 174; function
of, 610.

Thymus-nucleic acid, 281, 285.

Thyreoglobulin, 132, 608.

Thyroid gland, 604-609.

Tissue, o.steoplastic, 374; osteoidal, 374.

Tissue-extracts, 10.

Tissue-fat, calorific value, 333.

Tissue-juices, influence on coagulation of
blood, 540.

Toad poison, 690.

Toad-stools, 114.

Toluene, 243.

Toluric acid, 243.

Toluene diamine, 570.

Toluic acid, 243, 458.

Tonus, vascular, 437.

Tooth-structure, 493.

Tooth-wort, albumin crystals from, 122.

Toipedo marmorata,, 83.
oscellata, 83.

Toxines, 682-690.

Toxophor groups, 684.

Tracha?, 411.

Tradescentia, 459.

Tragacanth-gum, 24.

Transformation of nutriment albumin
into body albimiin, 209 et seq ; car-
bohydrate and protein, 305 el seq. ; fat
and carbohydrate, 103, 303 et seq. ; fat
and protein, 305, 323 et seq.

Transudates, 577, 578.

Trehalose, 37.

Tribrom-phenol, 252.

Trichlor-lactic acid, 278.
Triglycine, 185.
Triglycyl-glycine, 474.
Triglycyl-glycine-ester, 474.
Trihydroxyglutaric acid, 28.
Trimethylamine, 113, 114.
Trimethylamino acetic acid, 114.

722

SUBJECT INDEX.

TrimethyI-2-, 6-, dioxypurine, 1-, 3-, 7-
(caffein), 280.

Trimethylcarbinol, 34, 229.

Trimethyl-hydroxy-ethyl-ammoniuni hy-
droxide, 113.

Trimetliyl-vinyl-ammouium hydroxide,
114.

Triolein, 102.

Trioses, 19.

Trio-xypurin (2-, 6-, 8-), 238, 280.

Tripalmitin, 102. _

Trisaccharide.s, 40.'

Tristearin, 102.

Triticonucleic acid, 23.

Triton (Salamander), 674.

Tromnier's test for sugar, 25.

Trout, protamine from, 137.

Trypsin, 208, 482, 522.

Trypsin digestion, 163, 164, 189.

Trypsinogen, 522.

Tryptophane, 152, 251, 258, 260.

Tubulus contortus, 581.

Typhoid, 679.

Tryosine, 151, 202, 251, 254, 255, 256,
259, 272, 304.

Tryosinase, 447.

Tyrosyl-glycine, 1-, 187.

U.

Umbilical cord, 143.

Uracil, 281, 298.

Ur«mia, 595.

Uraminobenzoic acid. See carbamino-
benzoic acid.

Uraminoisethionic acid. See carbamino-
i.sethionic.

Uranium salts, cause of glucosuria, 81.

Urate of sodium, acid and neutral, 299,
593

Urea, 155, 225-227, 238, 278, 279, 295,
325; role in solution of uric acid
594; formation of, 228 et seq.; oxi-
dation of amino acids, 234; conju-
gation with, 234, 457; behavior to red
corpuscles, 550.

Urease, 482.

Uric acid, 236 et seq.; 277, 289; exogenous
and endogenous, 291 ; metabolism,
291; solubility of, 298, 593; elimi-
nation from kidneys, 586 et seq.

Uric acid diathesis, 298.

Uricolytical ferments, 294.

Urinary tubes, function of, .582.

Urine, 579 et seg. ; theory of elimination,
583, 586; composition of, 589, 590;
reaction of, 591.

Urobilin, 572.

Urochloralic acid, 34.

Urochrome, 572.

Uroerythrin, 572.

Uroferric acid, 270.

Uroleucic acid, 257, 271.

Uroxanthic acid, 271.

Ursocholeic acid, 516.

Utilization of foodstuffs, 650 et seq.
Utricularia, 168.

V.
Valeric acid, from celulose, 63.
Valeric aldehyde, d-, 149.
Value of foodstuffs, 333, 334, 336.
Vampyrella spirogyrse, 533.
Vanillin, oxidation in diabetes, 94.
Variations, individual, 8.
Vascular tonus in high altitudes, 436.
Vegetable acids, 56; oxidation of the

alkali salts in diabetes, 93.
Vegetable diet, 649-653.
Vegetable mucilages, 42.
Vegetarianism, 649-652.
Vignin, 653.
Vitellin, 134.
Vitelloses, 165.
Volemitol, 29.
Vorticella, 46, 52.

W.
Water, 53, 349, 354; influence on gastric

secretion, 500.
WTiale (sperm), 102.
Wheat, ash of, 369, 370, 380; gliadin and

gluten casein, 132.
Wheat-bran, 381.
Wlieat-flour, 380.
White-bread, iron and lime content, 370,

377, 380.
White-horse melanin, 145.
White of egg, composition of, 659.
White-snapper, protamine from, 137.
Wild raspberries, iron and lime content,

370.
Wild strawberries, iron and lime content,

370, 381.
Woman's milk. See Human milk.
Wood, pentosans from, 24.
Wool-fat, 116.
Work, heat equivalent of, 335, 336, 624,

625.

X.

Xanthine, 280, 297.

Xantho-proteic reaction, 162, 163.

Xanthorhamnin, 40.

Xylan, 24.

Xylitol, 28.

Xylol, 243, 458.

Xylose, ;-, 21, 24, 3.3, 282.

Y.

yea.st, 27, 47, 141.

Yeast-nucleic acid, 21, 22, 281.

Yeasts, expressed liquor from, 464.

Yeasts, preserved, 464.

Yolk of the hen's egg. See Egg-yolk.

Yolk-plates, 123.

Z.
Zein, 133, 173, 653.
Zymase, 464.
Zymogen, 205, 463, 465, 497.

5^ '^^\

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